Epitope Mapping by Antibody Competition

Methodology and Evaluation of the Validity of the Technique

Socrates J. Tzartos

1. introduction Most epitope mapping techniques can be classified into two groups: those

that use antigenic fragments (subunits, peptides, and so forth) and the specific antibody-binding regions are directly determined, and those that use intact antigen and the epitope determination is usually indirect.

Recently, it has become possible to localize epitopes in fine detail by the use of synthetic peptides of various kinds. Some of the recent synthetic and recom­binant peptide techniques allow the simultaneous production of many peptides, and make it possible to identify each contributing amino acid in an epitope (J, 2). The question therefore arises whether we still require techniques using intact antigens, which only permit indirect and approximate localization.

However, peptide mapping techniques are incomplete, since:

1. The fact that a monoclonal antibody (MAb) binds to a peptide corresponding to a sequential segment of an antigen does not guarantee that it also binds to that same sequence in the intact molecule. The peptide may simply mimic an irrel­evant conformational epitope on the antigen (perhaps formed from a number of distant segments), or the sequence that is identical to that of the synthetic peptide may be hidden or have an altered conformation in the intact molecule.

2. Many MAbs do not bind to any synthetic peptide, probably because their epitope is conformation-dependent.

3. Even when it is unequivocally established that the peptide corresponds to the actual epitope, its relative location on the intact molecule will remain unknown, unless the crystallographic structure of the molecule has been determined.

From Methods In Molecular Biology, vol. 66. Epitope Mapping Protocols Edited by G E Morris Humana Press Inc , Totowa, NJ

55

56 Tzartos

We have perfonned extensive competition experiments between many MAbs directed against the acetylchohne receptor (AChR) (3-6), as well as between MAbs and the natural antibodies in human anti-AChR sera (7), and have compared the data obtained in this way with those from detailed peptide mapping experiments (8—14). Overall, the two approaches confirmed and complemented each other, and showed the antibody-competition approaches to be surprisingly accurate.

Figures 1 and 2 compare the results of competition experiments between MAbs directed against sites on the cytoplasmic region of the AChR a- and P-subunits (5) with the subsequently derived fine mapping results from antibody binding to synthetic peptides (derived from the a- and p-subunit sequences) attached to polyethylene rods (9,10) according to the Pepscan technique of Geysen (1,2), as well as with earlier peptide mapping experiments using con­ventional larger peptides (8,13,14). In interpreting competition results, it should be taken into account that when two epitopes overlap, or even when the areas covered by the arms of two MAbs overlap, competition should be almost com­plete and mutually crosscompetitive. Thus, only marked mutual cross-competition should be taken as unequivocal evidence of overlapping epitopes, since weak or one-way inhibition may simply reflect a decrease in affinity owing to steric or allosteric effects. Therefore, we completely ignored cases of weak inhibition (<25%) and essentially only considered inhibition of >50%, paying particular attention to those antibodies giving inhibition values of >70%. It was shown that only those MAbs that bind to very close, or overlap­ping, sequential epitopes effectively show strong mutual crossinhibition of binding to the antigen. Interestingly, MAbs whose epitopes are separated by as few as six amino acids (anti-a MAb 8 vs MAb 142 in Fig. 1) did not signifi­cantly crosscompete. Similarly, in studies using anti-6-subunit MAbs, antibod­ies to sequential epitopes separated by only five amino acids showed only partial crosscompetition (12). Furthermore, differences between MAbs directed against overlapping epitopes could also be clearly detected by their differential competition pattern with other MAbs: e.g., anti-a-subunit MAbs 8 and 147, which bind to overlapping epitopes, showed complete mutual crosscompeti­tion, but only one, MAb 147, competed with MAbs 142 and 3 (Fig. 1). Simi­larly, the binding of anti-p-subunit MAb 117 to the AChR was inhibited by all MAbs directed against the overlapping epitope VICE-P, but MAbll7 itself could only weakly inhibit the binding of the same MAbs when used as the first (protecting) MAb (Fig. 2). A similar pattern was also seen for several MAbs to the 5-subunit (12).

It should, however, be noted that these MAbs were derived from animals immunized with SDS-denatured AChR and were selected using intact AChR. Because of this, we probably selected for MAbs directed against antigenic sites

Epitope Mapping by Antibody Competition 57

Protecting^'73 gl87 soluble mAbs / - • Segment of '

3 147 153 164 142 \ 8 155

a -subunit -ENKIFAODIDISDISGKQVTGEVIFQTPLIKNPOVKSAIEGVKY 3 i O 3S.0 310

Fig. 1. Mapping the binding sites of MAbs against the AChR a-subunit by compe­tition between pairs of MAbs for binding to the intact Torpedo AChR and comparison with their identified sequential epitopes by synthetic peptides. '^^I-a-bungarotoxin-labeled AChR was preincubated with a protecting soluble MAb and the complex was incubated with a Sepharose-bound MAb. The Sepharose-bound radioactivity was mea­sured and the percentage inhibition of binding owing to the protecting, soluble MAb was estimated. Large bars denote effective competition (modified from ref 5). Hori­zontal bars at the bottom represent the epitopes of the corresponding MAbs identified using synthetic peptides (9,10,14). The epitope for MAb 173 has not yet been deter­mined. The epitope for MAb 19 was only approximately localized by the use of large conventional synthetic peptides (8). Its exact epitope may form only part of the indi­cated a346-364 peptide and is probably located at its N-terminal end, as judged from the competition pattern with the other MAbs. The epitopes of the other MAbs have been accurately determined using Pepscan peptides. MAb 149 is an IgM, the large size of which may explain its broader protecting capacity.

whose conformation is virtually the same in the two states. This may be par­tially the reason for the very good correlation between the results for peptide mapping and antibody competition. Nevertheless, another large group of anti-AChR MAbs, derived from animals immunized with intact AChR, showed complete mutual crosscompetition for binding to the AChR (3,6) permitting us to define the main immunogenic region (MIR) of the AChR; several of these anti-MIR MAbs could be mapped by synthetic peptides, and all were found to bind to the same epitope, residues 67-76 of the a-subunit (8).

58 Tzartos

<J) n < E

8 n s o to s: a. <D c/i

172

125

11

94

112

111

148

11H

i?a

117

169

Protecting 169117 118 1t1 ,123 148 112 11

soluble mAbs Segment of /5-subunit

/}336 /3469

Fig. 2. Mapping the binding sites of anti-P-subunit MAbs on the intact Torpedo AChR by competition expenments (modified from ref 5) and comparison with pep­tide mappmg experiments. The experimental conditions were as in Fig. 1. The large horizontal bar at the bottom represents the region P336-469, and the small bars above it mark the locations of the sequential epitopes. The locations of the epitopes for MAbs 169 and 172 have only been approxhnately identified using proteolytic peptides (13), whereas the epitopes for the remaining MAbs have been accurately determined using Pepscan syn­thetic peptides (10). VICE-p, very immunogenic cytoplasmic epitope on p-subunit.

2. Materials 1. PBS: 0.9% NaCl, 10 mM sodium phosphate, pH 7.2. 2. PBS-Tween: PBS, containing 0.05% Tween-20. 3. PBS-bovine serum albumin (BSA): PBS, containing 1% BSA (see Note 1) and

0.02% sodium azide. 4. 0.IM sodium bicarbonate buffer, pH 9.6. 5. Anti-Ig produced by immunization of rabbits with the relevant Ig.

3. Methods The first antibody to be incubated with the antigen will be referred to as the

"first," or "protecting," antibody, although that which is incubated with the formed antigen-first antibody complex will be referred to as the "second" antibody. For best resuhs, care should be taken that the protecting antibody is present in excess over the antigen, whereas the amount of second antibody

Epitope Mapping by Antibody Competition 59

should be just sufficient to saturate the antigen (or about 80% of it). Tech­niques will be described for competition between antibodies from the same species or between antibodies from two different species. In general, one of the three reactants (the antigen and the two antibodies) is labeled by radioactivity or by conjugation to an enzyme. In some cases, indirect labeling can be used, i.e., labeled molecules are used that bind specifically to only one of the reac­tants (e.g., a ligand of the antigen, an anti-antibody, protein A, and so forth). Usually the assays are solid-state. Therefore, one of the nonlabeled reactants is immobilized on plastic wells or on Sepharose beads. Antibody competition mainly involves MAbs, but competition between MAbs and polyclonal sera may also be required to determine the percentage of serum antibodies directed against specific epitopes or regions (7).

3.1. Competition Between Homologous Antibodies 3.1.1. Labeled Antigen, Immobilized Second Antibody

A major advantage of this technique is that a single labeling step (that of the antigen) makes it possible to carry out crosscompetition experiments using anti-AChR antibodies of any type. It does, however, require that the second anti­body preparations be either purified or antibody-enriched {see Note 2).

3.1.1.1. DETERMINATION OF THE APPROPRIATE CONCENTRATION

OF LABELED ANTIGEN {SEE NOTE 3)

1. Add 50-nL samples of 20 |j,g/mL of second antibody in 0. IM sodium bicarbonate buffer, pH 9.6 (or in PBS) to the wells of a 96-well microplate (18-24 wells/ antibody), and incubate for 2 h at room temperature or overnight at 4°C {see Note 4) Among the antibodies tested, at least one "control" nonbinding anti­body should be included in order to determine background binding. Alternatively, 20 |ug/mL of BSA may be used as a negative control.

2. Wash the wells three times with PBS-Tween. 3. Fill the wells completely with PBS-BSA, and incubate for 30-60 mm at room

temperature. 4. Wash twice with PBS-Tween. 5. Place 50 |.iL of ' ^I-labeled antigen (20,000 cpm) alone, or mixed with various-

fold-excesses (e.g., 0, 1, 2, 4, 8, 16, 32, 64 or 0, 1, 3, 9, 27, 81) of unlabeled antigen in PBS-BSA in each well.

6. After 2 h incubation at 4°C, remove nonbound antigen by four washes with PBS-Tween.

7. Release the bound radioactivity by adding 100 \xL of 1% SDS, place in test tubes, and count on a y-counter.

8. After subtracting the background counts, plot the results (bound radioactivity vs antigen concentration) in order to select the proper antigen concentration for the competition experiments. Normally, the plot should show a plateau of bound radioactivity, which then starts to decrease. The antigen concentration at the point

60 Tzartos

at which the values start to fall is considered equimolar to the amount of active antibody on the plate. A value of 8(V-100% of this concentration is used in the subsequent steps (see Note 5).

3.1.1.2. DETERMINATION OF THE REQUIRED CONCENTRATION

OF FIRST (SOLUBLE) ANTIBODY

In this part, competition is between different samples of the same antibody (immobilized and soluble) using varying concentrations of the soluble antibody.

1. Repeat steps 1-4 of Section 3.1.1.1. 2. Preincubate the antigen at the predetermined concentration for 2-4 h at 4°C with

increasing concentrations of the first antibody in PBS-BSA 3. Place the mixture in the coated wells (50 |xL/well). 4. Repeat steps 6 and 7 of Section 3.1.1.1. 5. Plot the results (bound radioactivity vs concentration or dilution of the soluble

antibody used). For the subsequent competition experiments between heterolo­gous antibodies, whenever possible, a 10-fold excess of the soluble antibody (cal­culated as 20 times the concentration required to bind 50% of the plateau value [maximum bound radioactivity]) is chosen (see Note 6).

3.1.1.3. FINAL COMPETITION EXPERIMENT BETWEEN DIFFERENT ANTIBODIES

1. Repeat steps 1-4 of Section 3.1.1.1. for coating, washes, and blocking. 2. Preincubate the previously determined concentrations of labeled antigen and pro­

tecting antibodies (including controls with a nonbinding antibody or without anti­body) for 2-4 h as above.

3. Add 50 |iiL of the mixture to each washed well, and incubate for 2 h at 4°C. 4. Repeat steps 6 and 7 of Section 3.1.1.1.

A variation to this technique, in which the second antibody is immobilized on Sepharose beads rather than on ELISA plates, is described in Note 7.

3.1.2. Labeled Second Antibody, Immobilized Antigen

The various antibodies to be tested as "second antibodies" must be rela­tively pure (50% "pure" is adequate), and either radiolabeled (preferably by ^ I) or peroxidase-conjugated (see Note 3).

3.1.2.1. COATING WITH THE ANTIGEN

1. Add 50-|iL samples of 20 |ig/mL (or less: 1-10 |.ig/mL) of antigen in 0. lAf sodium bicarbonate buffer, pH 9.6 (or in PBS) to the wells of a 96-well microplate, and incubate for 2 h at room temperature or overnight at 4°C. If the antigen is a mem­brane protein, which requires the presence of detergent in the buffer, solubilize it in the appropriate concentration of the required detergent, and then dilute it in a buffer lacking detergent (PBS or sodium bicarbonate) to 20 jig/mL or less for the coating step (a final concentration of 0.05% Triton X-100 is acceptable). An equal concentration of BSA may be used as a negative control

2. Repeat steps 2-4 of Section 3.1.1.1.

Epitope Mapping by Antibody Competition 61

3.1.2.2. DETERMINATION OF THE REQUIRED ANTIBODY CONCENTRATIONS

1. Add 50-jxL samples, containing 20,000 cpm of labeled antibody (mixed with 0, 3-, 10-, or 30-fold excess of the same unlabeled antibody in PBS-BSA) to each well, and incubate for 2 h at room temperature.

2. Wash the plates four times with PBS-Tween. 3. Measure the bound radioactivity and plot the results (bound radioactivity vs

amount of antibody used) as in steps 7 and 8 of Section 3.1.1.1. Normally, the plot should start with a plateau that suddenly starts to decrease. The antibody concentration at the end of the plateau (when it starts decreasing) is considered equimolar to the active antigen on the plate.

4. Repeat steps 1 and 2 in Section 3.1.2.1.

3.1.2.3. COMPETITION EXPERIMENT

1. Add 50 pL of unlabeled first antibody (10 times the above determined "equimo­lar" concentration) in PBS-BSA to the antigen-coated wells, and incubate for 3-4 h at room temperature.

2 Add (without removal of the unlabeled antibody) 50 \xL of '^'l-labeled second antibody (if necessary, mixed with unlabeled antibody to give the "equimolar" concentration) in PBS-BSA, and incubate a further 2 h at room temperature.

3. Wash the plates and count the radioactivity as in steps 2 and 3 of Section 3.1.2.2. (see Note 6).

3.2. Competition Between Antibodies from Two Different Species

Competition between antibodies from different species has an additional advantage. Using anti-Ig sera, which bind selectively to Ig from the species in which the second antibody was raised (e.g., of human origin), the competition test can be performed totally in solution with the only labeled species being the antigen. For example, we often perform competition experiments between rat MAbs and human sera for binding to the AChR.

1. Label the antigen as m Section 3.1.1. 2. Obtain antiserum specific for Ig from the species in which the second antibody

was raised (e.g., human sera: use antihuman Ig). 3. If this antiserum partially crossreacts with the first antibodies, preincubate it for

at least 3 h with normal serum fi-om the corresponding first species, e.g., rat (the appropriate amounts have to be determined experimentally, but in my laboratory we mix 1 mL of anti-Ig serum with 50 |:iL of normal serum) and centrifuge to eliminate any aggregates formed.

4. Add an additional 50 nL of normal serum and incubate for at least 2 h, usually without any further pellet becoming visible. The pretreated antiserum must then be tested to verify that it does not bind the protecting antibodies and to determine the amount needed to precipitate the second antibodies, as follows.

62 Tzartos

5. Incubate 20 |jL of PBS-BSA containing 20,000 cpm (10-100 fmol) of labeled antigen with 20 juL of PBS-BSA containing a 10-fold (or greater) molar excess of protecting first antibody or an equimolar amount of second antibody (or 80% of this). To ensure that a significant pellet will be formed in the subsequent steps, the 20 nL of antigen should also contain 0.1 jxL of normal serum from the species in which the second antibody was raised to act as a carrier.

6. After 3 h of incubation, add 20-nL dilutions (from undiluted to 1/10) of the pre-treated anti-antibody, and incubate for 1 h.

7. Add 1 mL of washing buffer and mix. 8. Centrifuge for 3-5 min and wash the pellets one to two times. 9. Count the radioactivity of the pellets.

The results should (1) show that the tubes with the protecting antibody con­tain only background level of radioactivity, and (2) determine the appropriate dilution of anti-antibody for the subsequent experiments.

3.2.1. Final Experiment 1. Incubate 20 |xL of PBS-BSA containing about 20,000 cpm (10-100 fmol) of

labeled antigen plus 0.1 mL of normal serum from the species in which the sec­ond antibody was raised, for 3 h with 20 \xL of PBS-BSA containing a 10-fold molar excess of "protecting" antibody.

2. Add 20 |LiL of a sample containing second antibody equivalent to about 80% of the antigen used, and incubate for a further 2 h.

3. Add 20 |.iL of a sample containing the above predetermined dilution of pretreated anti-Ig, and incubate for 1 h.

4. Add 1 mL of washing buffer, mix, and centrifuge. 5. Wash the pellets one to two times, and count their radioactivity (see Note 8).

Alternatively, by using enzyme-conjugated antiserum specific for the Ig of the second species, the competition can be performed as a solid-phase assay without any requirement to label any of the three reactants: antigen, first, and second antibody (see Note 9).

4. Notes 1. Some investigators use 3% BSA; in my laboratory we have not found it signifi­

cantly better, but we found it significantly more expensive. An efficient eco­nomical alternative is the use of 3% powdered milk instead of BSA

2. Ammonium sulfate precipitates of ascites fluids are sufficiently pure, but hybri-doma culture supematants containing serum are not appropriate. However, an easy means of obtaining sufficiently pure antibody preparations is to culture the hybridomas for 24 h in serum-free medium (DMEM); under these conditions, in our experience, the cells continue to produce antibody at about one-third of their normal rate. This can then be concentrated by ultrafiltration (e.g., by Amicon, Beverly, MA), but not by ammonium sulfate precipitation, since the protein con­centration is too low.

Epitope Mapping by Antibody Competition 63

3. The antigen that is routinely used in my laboratory, the AChR, is easily indirectly labeled by preincubation with ' ^I-labeled a-bungarotoxin, which binds very strongly in the region of the acetylcholine-binding sites. It is, therefore, a simple process to label AChRs without the need for purifying them. Should such a method not prove suitable, the antigen can be conjugated to peroxidase or labeled directly with '2^1, using the chloramine-T or other methods (15). The use of the radiola­beled antigen is described below, but the method can be easily modified for use with peroxidase-conjugated antigen. If the concentration of the unlabeled antigen is known, a reasonable estimate of the molarity of the labeled antigen can be made assuming about 20-30% loss during the labeling and purification procedures. How­ever, it is probable that a large percentage may be inactivated during labeling.

4. If necessary, much lower concentrations of antibody can be used (diluted just before use), but the incubation times should be increased (e.g., 4 and 6 h for 4 and 1 |ig/mL of antibody, respectively).

5. If the amount of bound radioactivity is low and does not exhibit a plateau, this may mean either that the amount of active antibody is low or that the specific radioactivity of the antigen is very low. The experiment must be repeated using 5000,10,000, and 20,000 cpm of antigen without unlabeled antigen. If the pla­teau is very low, this may mean either that the affinity of the antibody-antigen interaction is very low or that the radioactive antigen has been damaged, possibly during labeling. In this case, if the background is much lower, the experiment may be repeated with the use of much more radioactivity. If the whole plot is a high plateau, the experiment must be repeated using antigen mixtures with higher amounts of unlabeled antigen. Alternatively, the concentration of the coating antibody may be decreased to 0.1-1 |Lig/mL, so that lower amounts of labeled antigen can be used.

6. To ensure fiirther that the first antibody gives sufficient protection even when its affinity for the antigen is much lower than that of the second antibody, it can be tested at varying degrees of excess (e.g., 5- to 50-fold excess); their effect should not be very different; otherwise, even higher excesses may be needed.

7. Second antibody immobilized on Sepharose beads: This method has the advan­tage that immobilized antibodies can be prepared in large quantities and remain active for more than a year, which obviously improves the reproducibility of results. In addition, there is not the strict necessity for the antibody preparation to be purified before immobilization, since CNBr-activated Sepharose beads have a high protein-binding capacity. Nevertheless, the method does have significant disadvantages, such as the need to use a large number of test tubes (and many tedious washes) instead of a single multiwell plate. The work can be reduced by the use of flexible polyvinyl chloride V-bottom multiwell plates (Dynatech Labo­ratories), which are washed by centrifugation in special rotors and the wells indi­vidually cut out to measure the bound radioactivity. If enzyme labeling is used instead of radiolabeled antigen, the colored supematants must be transferred to ELISA plates for measurement. I would recommend this approach only in con­junction with the use of radioactive antigen.

64 Tzartos

a. Antibody immobilization on CNBr-Sepharose beads: This is performed according to the manufacturer's instructions. However, since the use of large Sepharose beads is inconvenient, in my laboratory we break them up into small pieces by sonication just before conjugation.

b. Titration of immobilized antibody: The titers of the immobilized antibodies are estimated in order to determine the amount of immobilized antibodies and of radioactive antigen to use. Make 1/5,1/15, 1/50, and 1/150 suspensions of the Sepharose-antibody beads in PBS-BSA, and incubate (with shaking), in Eppendorf tubes, 50 |iL of suspension and 50 )LIL of labeled antigen (20,000 cpm ' ^I-labeled antigen plus increasing concentrations of unlabeled antigen as above) for 2-4 h at room temperature. Wash two to three times (brief cen-tnfugation) with 1 mL of washing buffer (we use PBS-0.5% Triton X-100, which is suitable for AChR solubilization), and count the remaining radioac­tivity Plot the data (preferably bound radioactivity vs antigen used), and select an appropriate pair of antibody-antigen concentrations: Choose the minimum amoimt of Sepharose-antibody that shows a plateau and a corresponding anti­gen concentration just after the plateau (i.e., equimolar concentration).

c. Final steps; The methodology used to determine the required concentration of first antibody and for the final competition experiments between different antibodies will be obvious from the above information.

8. Similar differential precipitation of labeled second antibody may be applied to competition experiments between antibodies from single species when only one of the antibodies binds protein A (or protein G). Sepharose-immobilized protein A (or G) can replace the precipitating anti-Ig, whereas '- I-labeled or peroxidase-conjugated protein A (G) can replace the labeled anti-Ig. In such cases, however, an antibody may be used only as second or first antibody depending on whether or not It binds protein A (or protein G).

9. Heterologous antibody competition in solid state (ELISA or RIA): Only the prin­ciple of the technique will be described here, since the details will be evident from the previously described techniques. Any of the three reactants may be immobilized in the wells. If the second antibody is to be immobilized, the proce­dure is the following: a. The second antibody is plated on the microwells. b. After washes, a preincubated mixture of predetermined amounts of the anti­

gen and an excess of first antibody are added and further incubated. c. After washes, peroxidase-conjugated anti-Ig specific for the species of the

first antibody (preabsorbed with normal serum from the species of the second antibody) is added, and the mixture is incubated. This is followed by washes, addition of substrate for color development, and absorbance measurement.

Acknowledgments This work was supported by grants to S. J. T. from the Association Francaise

contre les Myopathies, the BIOMED-1 program of EC (BMH1-CT93-1100), and the Human Capital and Mobility program of EC (CHRXCT94-0547).

Epitope Mapping by Antibody Competition 65

References 1 Geysen, H. M., Meloen, H. R., and Barteling, S. J. (1984) Use of peptide synthe­

sis to probe viral antigens for epitopes to a resolution of a single amino acid. Proc. Nad. Acad. Set. USA 81, 3998-4002.

2. Geysen, H. M., Tainer, J. A., Rodda, S. J., Mason, T. J., Alexander, H., Getzoff, E., and Lemer, R. A. (1987) Chemistry of antibody binding to a protein. Science 235,1184-1190.

3. Tzartos, S. J., Rand, D. E., Einarson, B. E„ and Lindstrom, J. M. (1981) Mapping of surface structures of Electrophorus acetylcholine receptor using monoclonal antibodies. 7. Biol. Chem. 256, 8635-8645.

4. Tzartos, S. J. and Kordossi, A. (1986) Acetylcholine receptor conformation probed by subunit-specific monoclonal antibodies, in Nicotinic Acetylcholine Receptor, NATO ASI series vol. H3 (Maelicke, A., ed.), Springer-Verlag, Heidel­berg, pp. 35-47.

5. Kordossi, A. and Tzartos, S. J. (1987) Conformation of cytoplasmic segments of acetylcholine receptor a and b subunits probed by monoclonal antibodies. Sensi­tivity of the antibody competition approach. EMBOJ. 6, 1605—1610.

6. Kordossi, A. A. and Tzartos, S. J. (1989) Monoclonal antibodies against the main immunogenic region of the acetylcholine receptor. Mapping on the intact mol­ecule. J. Neuroimmunol. 23, 35-40.

7. Tzartos, S. J., Seybold, M., and Lindstrom, J. (1982) Specificities of antibodies to acetylcholine receptors m sera from myasthenia gravis patients measured by monoclonal antibodies. Proc. Natl. Acad. Sci. USA 79, 188-192.

8. Tzartos, S. J., Kokla, A , Walgrave, S., and Conti-Tronconi, B. (1988) Localiza­tion of the main immunogenic region of human muscle acetylcholine receptor to residues 67-76 of the a-subunit. Proc. Natl. Acad. Sci. USA 85, 2899-2903.

9. Tzartos, S. J. and Remoundos, M. S. (1992) Precise epitope mapping of mono­clonal antibodies to the cytoplasmic side of the acetylcholine receptor a-sub­unit. Dissecting a potentially myasthenogenic epitope. Eur. J Biochem. 207, 915-922.

10. Tzartos, S. J., Valcana, C, Kouvatsou, R., and Kokla, A. (1993) The tyrosine phosphorylation site of the acetylcholine receptor p-subunit is located in a highly immunogenic epitope implicated in channel function. Antibody-probes for p sub-unit phosphorylation and function. EMBOJ. 12,5141-5149.

11. Tzartos, S. J., Tzartos, E., and Tzartos, J. S. (1995) Monoclonal antibodies against the acetylcholine receptor y-subunit as site specific probes for receptor tyrosine phosphorylation. FEBSLett. 363,195-198.

12. Tzartos, S. J., Kouvatsou, R., and Tzartos, E. (1995) Monoclonal antibodies as site-specific probes for the acetylcholine receptor 8-subunit tyrosine and serine phosphorylation sites. Eur. J. Biochem. 228,463-472.

13. Ratnam, M., Sargent, P., Sarin, V., Fox, J. L., Le Nguyen, D., Rivier, J., Criado, M., and Lindstrom, J. (1986) Location of antigenic determinants on primary sequences of the subunits of the nicotinic acetylcholine receptor by peptide map­ping. Biochemistry 25,2621-2632.

66 Tzartos

14. Ratnam, M., Le Nguyen, D., Rivier, J., Sargent, P., and Lindstrom, J (1986) Transmembrane topography of the nicotinic acetylcholine receptor: immu­nochemical tests contradict theoretical predictions based on hydrophobicity pro­file. Biochemistry. 25, 2633-2643.

15. Harlow, E. and Lane, D. (eds.) (19SS) Antibodies, A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.