Patent Application: US-31820094-A

Abstract:
this invention is directed towards peptidic compositions , methods , and diagnostic kits for the accurate and sensitive detection of human acetylcholine receptor autoantibodies associated with the disease myasthenia gravis . eighteen synthetic overlapping oligopeptides encompassing the entire extracellular domain of the α - chain of human achr and an additional peptide corresponding to the extracellular connection between the two transmembrane regions were prepared . the immunologic reactivity of these peptides against autoantibodies in the plasma of patients with mg was ascertained by solid - phase radioimmunoassay . autoantibody responses were subjected to genetic regulation as indicated by the variation in recognition profiles from patient to patient . however , it was possible to detect achr autoantibodies in a heterogenous patient population by employing a peptide mixture comprising at least four peptides . these peptides correspond to the following regions of the achr : α12 - 27 , α111 - 126 , α122 - 138 , and α182 - 198 . these reagents provide a peptide - based direct antibody binding method for the detection of myasthenogenic autoantibodies .

Description:
in general aspects , certain protocols and procedures will be applicable to the various methods and compositions of matter of the invention . these general techniques are detailed below . the peptides used for certain particular examples of the invention are shown in fig1 and correspond to the sequence id nos . 1 - 6 . these peptides , as well as other peptides produced using the methods of the invention , were synthesized either by t - butyloxycarbonyl ( t - boc ) on a phenylacetamidomethyl ( pam ) resin or by 9 - fluorenmethylcarbonyl ( fmoc ) amino acids on a benzyloxybenzyl alcohol resin as described elsewhere ( mccormick and atassi 1984 ; mulac - jericevic and atassi , 1987 ; atassi et al ., 1991 ), together with methods for purification and characterization of the peptides . other synthetic procedures can be utilized as well so long as the synthesis results in biologically active peptides . to prepare the succinate esters of mpeg and pva , one gram of mpeg ( molecular weight , 5000 ) or pva ( molecular weight 3 , 000 ) is dissolved in 5 ml anhydrous pyridine at 50 ° c ., and to these solutions aliquots of succinic anhydride ( 0 . 5 g each ) are added as a dry powder at 1 - hour intervals . following the last addition , the reaction mixture is stirred for 2 hours at 50 ° c ., after which it is evaporated to dryness on the flash evaporator . the residue is dissolved in water and evaporated to dryness and this washing with water on the evaporator is repeated several times until the odor of pyridine in the residue is very faint . the residue is dissolved in water ( 10 ml ) and dialyzed , in a 1000 - molecular weight cut - off dialysis membrane , against several changes of distilled water and finally freeze - dried ( yield 0 . 92 - 0 . 95 g ). complete succinylation of the polymers is confirmed by a negative reaction for hydroxyl and a positive reaction for carboxyl groups . determination of hydroxyl groups is carried as follows . a solution of test sample ( 365 μl containing 2 mg of mpeg , pva mpeg - su , pva - su , succinic anhydride , and standards containing various amounts of methanol from 0 to 1 . 5 μmole in 0 . 06m sodium phosphate buffer ph 7 . 5 , containing 0 . 09m nacl , is mixed with 182 μl of 0 . 75m perchloric acid . a blank is prepared which contains the same reagents but without a test sample . after mixing , the tubes are centrifuged ( 2000 rpm , 20 min .) and 365 μl aliquots from each tube are transferred to clean test tubes . to each tube is added 40 μl of 2 % kmno 4 ( in water ), the solutions are mixed for exactly 1 minute , then 40 μl of freshly prepared 10 % sodium sulfite ( in water ) is added and the tubes are immediately shaken vigorously . at this point , the solutions should become completely decolorized . to these solutions is added 1 . 45 ml of chromotropic acid reagent [ 8 mg of 4 , 5 - dihydroxy - 2 , 7 - naphthalene - disulfonic acid disodium salt ( sigma chemical co ., st . louis , mo .) dissolved in 0 . 10 ml h 2 o and 1 . 35 ml sulfuric acid solution ( conc . h 2 so 4 / water , 2 : 1 , v / v )]. the tubes are covered and placed in boiling water for 15 min . after which they are cooled to room temperature and the absorbance of the solutions is read at 580 nm against the blank solutions in the reference cell . the hydroxyl group content of a sample is determined based on the methanol standard curve of absorbance versus amount of methanol ( a 1cm 580 for 1 μmole of ch 3 oh = 0 . 856 ). carboxyl groups are detected by the following method . an aliquot of a water solution of the test sample ( 20 μl containing 1 mg of mpeg , pva mpeg - su or pva - su ) is applied as a spot on whatman no . 3mm chr paper . the spot is dried with cold air and the paper is then stained by spraying with a solution of 0 . 04 % bromothymol blue in ethanol , preadjusted to ph 8 . 0 with 0 . 2m boric acid . mpeg - su and pva - su give bright yellow spots on a blue background , whereas the spots of mpeg and pva appear blue . a coupling method for the preparation of mpeg - peptide and pva - peptide conjugates is illustrated in fig2 . the synthesis resin ( 0 . 1 g ) carrying the completed synthetic peptide ( 0 . 025 m mole ), with all the side chain protecting groups intact , is swollen in a synthesis vessel in methylene chloride overnight . the t - boc protecting group on the α - amino group of the synthetic peptide is removed by treatment with 40 % trifluoroacetic acid containing 2 % anisole and 2 % dimethylformamide ( dmf ) at room temperature for 30 minutes . for the fmoc - peptides , the n . sup . α - fmoc protecting group is removed by 20 % piperidine in dmf . for coupling of mpeg - su , a three - molar excess in 1 . 5 ml of dmf is reacted with 0 . 2 ml of 50 % dicyclohexylcarbodiimide ( dcc ) or diisopropylcarbodiiniide ( dipc ) in methylene chloride for 20 minutes , filtered to remove dicyclohexyl urea ( if dcc is used ), then added to the n - terminal - deprotected peptide - resin and allowed to react for 24 hours . for coupling of pva - su , larger reaction volumes are needed because of its tendency to gel . three - molar excess of pva - su is dissolved in 8 . 0 ml of dmf and to this is added 0 . 20 ml of 50 % dcc or dipc in methylene chloride . three cycles of recoupling of mpeg - su or pva - su to the peptide - resin are done . the complete blocking of the α - nh 2 is confirmed by a ninhydrin test ( kaiser et al , 1970 ). after the α - amino group on the peptide is completely blocked , uncoupled mpeg - su or pva - su is washed out of the vessel with methylene chloride and then methanol . the peptide conjugate is cleaved from the resin by hf ( sakakibara et al , 1967 ), if a pam resin and t - boc amino acids are used , or by treatment ( 2 . 5 hr ) with 55 % trifluoroacetic acid in methylene chloride if a benzyloxybenzyl alcohol resin and fmoc amino acids are used . any residual uncoupled peptide is removed from the conjugate by gel filtration on a sephadex g - 75 fine column ( 1 . 5 × 75 cm ) in 0 . 1m ammonium bicarbonate and by high pressure liquid chromatography ( hplc ) on a size exclusion column ( waters protein pack 60 , 0 . 7 × 30 cm ) which is eluted with 0 . 2m ammonium bicarbonate containing 20 % acetonitrile at a flow rate of 0 . 7 ml / min . the purification of torpedo achr is carried out as described elsewhere ( froehner , 1979 ; mulac - jericevic and atassi , 1987 ). briefly , the electric organ of torpedo californica ( pacific bio - marine laboratories , ca ) is homogenized and the membrane proteins are extracted in 1 % triton x - 100 ( sigma chemical company , mo ). after centrifugation , the achr in the supernatant is affinity purified on a cobratoxin sepharose cl4b column using 1m carbamylcholine in 1 % octyl β - d - glucopyranoside ( sigma chemical company , mo ) for the elution of the achr . the purified achr is composed of the expected four subunits ( α , β , γ , δ ) as demonstrated by sds - page ( laemmli , 1970 ). the structure of peptide α125 - 148 ( fig1 seq . id no . 5 ) of torpedo achr was based on the amino acid sequence of the extracellular part of the receptor ( noda et al ., 1982 ). the peptide was synthesized and purified as described by mccormick and atassi ( 1984 ). cyclization of the synthetic peptide α125 - 148 was performed under conditions previously described ( mccormick and atassi , 1984 ). the monomeric form was separated by gel filtration on sephadex g - 25 fine ( pharmacia fine chemicals ) column in 0 . 1m ammonium bicarbonate . after purification , homogeneity of the monomeric peptide was confirmed by high - voltage paper electrophoresis ( atassi and saplin 1968 ). the amino acid composition of the peptide was in excellent agreement with that expected from its sequence . a nonsense peptide , having a structure ( hfksfhsfsvsgetvfevteag ) totally unrelated to achr , was also synthesized and employed as a negative control . the coupling method for the preparation of mpeg - peptide conjugates is described above and in atassi and manshouri ( 1992 ). after synthesis of the conjugates and cleavage from the synthesis resin , any residual uncoupled peptide was removed from the conjugate by gel filtration on a column ( 1 . 5 × 75 cm ) of sephadex g - 75 fine in 0 . 1m ammonium bicarbonate . after lyophilization , the mpeg -( α125 - 148 ) conjugate was cyclized and the monomeric species isolated as described ( mccormick and atassi , 1984 ). a sample of the purified peptide - mpeg conjugate was confirmed to be free of the uncoupled peptide by high performance liquid chromatography ( hplc ) on a size exclusion column ( waters protein pack 60 , 0 . 7 × 30 cm ) which was eluted with 0 . 2m ammonium bicarbonate containing 20 % acetonitrile at a flow rate of 0 . 7 ml / min ( fig4 ). six weeks old c57 / bl6 mice were purchased from charles river breeding laboratories ( wilmington , mass .). preimmune sera were obtained from the mice for use as controls in subsequent antibody binding assays . after 2 - weeks rest , the mice were divided into three groups which received , at eleven , seven and three days before immunization with achr , an intraperitoneal injection ( 5 μg in 25 μl of pbs [ 0 . 15m nacl in 0 . 01m sodium phosphate buffer , ph 7 . 2 ]) of either the mpeg -( α125 - 148 ) conjugate ( group 1 ), mpeg - nonsense peptide conjugate ( group 2 ) or unaltered free peptide α125 - 148 ( group 3 ) ( fig5 ). then , on day 0 , the mice were immunized subcutaneously in one hind footpad and intramuscularly in the same side shoulder with 20 μg of torpedo achr in 100 μl emulsion containing equal volumes of complete freund &# 39 ; s adjuvant and pbs . thirty days later , the mice were immunized with a similar dose of the receptor in the opposite footpad and shoulder . on the 37th day , test bleeds were obtained from the mice for determination of the antibody titers . electrophysiological studies were performed on the 38th and 39th days . finally , the mice were sacrificed on the 40th day and lymph node cells were obtained for t cell studies . to document the electrophysiological evidence of eamg , amplitudes of serial muscle action potentials were measured by electromyography ( emg ) during the repetitive stimulation of the nerve in immunized mice , using the mystro emg system ( teca corporation ). a pair of wire electrodes were surgically implanted , encircling the sciatic nerve , two days before emg . the nerve was stimulated through the implanted electrodes by 3 hz trains of supramaximal electric current with a duration of 200 microseconds . the corresponding muscle action potentials were recorded with an electrode subcutaneously inserted over the gastrocnemius muscle . a reference electrode was placed at the ankle . ether inhalation was used during the surgical and recording procedures . the amplitude of the initial evoked potential ( p1 ) was compared to the third , fourth and fifth potentials , and the potential with the maximum amplitude deviation from the pi on either 3 hz or 5 hz stimulation was taken as ps . the change of the amplitude was calculated as follows : ## equ1 ## students &# 39 ; t - test was used to analyze the differences of amplitude changes between the two groups . a typical myasthenic response of a greater than 10 % decrement was considered to constitute an electrophysiological evidence of eamg . the frequency of electrophysiological eamg was calculated in each group and analyzed by chi - square test . one mg of the synthetic peptide was dissolved in 50 μl dimethylformamide and then diluted with pbs to 25 μg / ml . a solution of torpedo achr in pbs was prepared to contain 2 . 5 μg / ml . aliquots ( 50 μl / well ) of the peptide or achr solutions were added to a 96 - well microtiter plate ( falcon micro test iii flexible assay plate , becton dickinson , oxcard , calif .) and the plates were incubated at room temperature overnight . after washing 3 times with pbs , the plates were blocked with 0 . 25 % bovine serum albumin ( bsa [ bovine serum albumin ]) in pbs ( 50 μl / well ) at 37 ° c . for 90 minutes . the plates were washed again 5 times with pbs and to each well was added 50 μl of the serum from achr - immunized mice , pre - diluted with pbs containing 0 . 10 % bsa , and the plates were covered and allowed to stand at room temperature overnight . after 5 washes with pbs , 50 μl of rabbit igg antibodies ( 2 . 5 μg / ml ) against mouse igg and igm ( accurate chemical scientific corporation , westbury , n . y .) was added to each well and the plates were incubated at 37 ° c . for 3 hours . the plates were then washed 5 times with pbs and 125 i - labeled protein a ( 2 × 10 5 cpm in 50 μl of pbs - 0 . 1 % bsa ) was added to each well . the plates were incubated for 3 hours at room temperature , after which they were washed and the wells were cut out and counted for bound radioactivity . pre - immune serum samples , obtained from the mice prior to any experimental treatments , were used as controls to correct for non - specific binding . lymph node cells ( lnc ) were harvested from the achr - primed mice of each group ten or eleven days after the last achr immunization . the cells were suspended in rpmi 1640 ( gibco , grand island , n . y .) containing 1 % fresh autologous normal mouse serum . the number of viable cells was determined by vital staining with fluorescein diacetage . viable lnc ( 5 × 10 5 cells / well ) were co - cultured in triplicate in flat bottom microtiter plates with various concentrations of mitogen , antigen or synthetic peptide in a final volume of 200 μl per well . the antigens used were torpedo achr peptides ( α1 - 18 and α125 - 148 , in the dose range 10 - 40 μg / ml ), and torpedo achr ( dose range , 1 . 5 - 6 . 0 μg / ml ). lysozyme and ovalbumin ( 100 μg / ml ) and synthetic nonsense peptide ( essgtgiessgtgi , dose range 10 - 40 μg / ml ) were used as negative controls . concanavalin a ( 1 μg / ml ) and lipopolysaccharide ( 500 μg / ml ) were used as positive controls to monitor the viability of the cells . after incubation for 3 days at 37 ° c . in a humidified air / co 2 ( 19 : 1 ) atmosphere , the cultures were pulsed ( 18 hr ) with 1 μci / well [ 3 h ]- thymidine ( research products international , mount prospect , ill .) and then harvested on to glass microfiber filters ( whatman , clinton , n . j .) for counting of radioactivity by liquid scintillation . after purification , mpeg and pva peptide conjugates were homogeneous molecular species and were confirmed to be free of the uncoupled peptide by hplc on a size exclusion column ( see fig3 for an example ). furthermore , sequence analysis showed that the n - terminal was free in the uncoupled peptides and was completely blocked in the peptide conjugates . it should be noted that these peptides contained all possible amino acids . furthermore , the attachment of mpeg or pva to the n - terminal did not preclude the formation of intramolecular disulfide bonds in appropriate peptides ( peptides 5 and 6 in fig1 sequence id nos . 5 and 6 , respectively ). it is , therefore , clear that this reaction should be universally applicable to coupling mpeg or pva to any other synthetic peptide . the present invention allows the preparation of peptide conjugates to mpeg or pva by using a coupling reaction which ensured that the mpeg was linked to the peptide via its α - amino group on the n - terminal amino acid while the peptide is still attached to resin . this method provided a 1 : 1 , tail - to - head ( mpeg or pva to peptide ), monomeric conjugate of high purity . as noted previously , however , chemistries designed to mpeg derivatize the peptides of the invention at the carboxy terminus or at both the amino and carboxy termini are known to those of skill in the art and are expressly included within the scope of the present invention . all the amino acid side chains within the conjugate , except for the n - terminal , remained unaltered and , because they were not attached to mpeg or pva groups , they were capable of participating in immune recognition and epitope - specific immunoregulatory mechanisms . in contrast to the methods of the invention , mpeg - protein conjugates have been made by coupling mpeg to the protein , usually via ε - amino groups of lysine residues ( wei et al , 1981 ; nordvall et al , 1986 ; holford - strevens et al , 1987 ; jackson et al , 1987 ). this results in multiple substitutions on the protein surface and in modification of a number of lysine residues . the product would be expected to contain a mixture of molecular species of derivatives . the use of such compounds as tolerogens would potentially give rise to serious problems in reproducibility and efficacy , particularly in the recognition of individual epitopes by the immune system . the general approach of epitope directed immunosuppression by well - defined tolerogenic peptide conjugates offers useful refined strategies for modulation of antibody responses to immunopathogenic sites on multi - determinant complex protein antigens . because it provides a means to suppress the production of antibodies against the pathogenic epitopes , it should be applicable to autoantigens , alloantigens and allergens . mpeg itself does not seem to have any harmful or toxic effects in humans . allergens conjugated to mpeg have already been safely administered to human subjects suffering from asthma ( mosbech et al , 1990 ) and honeybee venom allergy ( muller et al , 1987 ). thus , the invention methods of mpeg or pva conjugation to peptides should prove important , if not essential , for the employment of these peptide conjugates in specific tolerance to epitopes of protein antigens . although some immunized mice showed muscle weakness , quantitative assessment of muscle weakness was difficult and , therefore , muscle action potentials were measured by emg . groups 1 , 2 and 3 consisted of 25 , 27 and 16 mice , respectively . additional 24 mice received no intraperitoneal injections or immunizations ( group 4 ). the typical myasthenic decremental response was reversible upon intraperitoneal injection of 250 μg edrophonium chloride ( fig6 ). the results are summarized in fig7 . none of the mice in group 4 showed a decremental response of the compound action potentials greater than 10 %, and their mean amplitude change was + 2 . 8 % ( i . e . 2 . 8 increment ). five ( 20 %) of the 25 mice in group 1 showed a decremental response greater than 10 %, while 13 ( 48 %) of the 27 mice of group 2 and 9 ( 56 %) of the 16 mice of group 3 developed the decremental response . the differences between group 1 and groups 2 or 3 were statistically significant ( p & lt ; 0 . 05 , chi - square test ), but were insignificant between group 2 and group 3 . the mean amplitude change in group 1 was - 5 . 4 % ( i . e . a 5 . 4 % decrement ), which was significantly smaller than that in group 2 (- 12 . 0 %; p & lt ; 0 . 05 ) or group 3 (- 15 . 9 %; p & lt ; 0 . 05 ), but greater than in group 4 (+ 2 . 8 %; p & lt ; 0 . 05 ). the results of antibody binding to achr and to selected regions of the e chain are summarized in fig8 . the antibodies bound to peptide α125 - 148 ( fig8 a ) were significantly decreased in the anti - achr antisera from the group of mice which received the mpeg -( α125 - 148 ) conjugate ( group 1 ) prior to immunization with torpedo achr , whereas mice that had received mpeg - nonsense peptide ( group 2 ) or free α125 - 148 ( group 3 ) prior to achr immunization exhibited no decrease in the antibodies directed against this region ( fig8 a ). antibodies bound to whole torpedo achr ( fig8 b ), to peptide α45 - 60 ( fig8 c ) and to peptide α182 - 198 ( fig8 d ) were not significantly different among the three groups ( p & gt ; 0 . 1 ). to further confirm the epitope specific suppression of the antibody response , serum samples of the mice within each group were pooled and used in serial dilutions for determining the levels of antibodies against the region α125 - 148 ( fig9 ). the antiserum mixture from group 1 [ pretreated with mpeg -( α125 - 148 )] had consistently lower amounts of antibody binding as compared to the antiserum mixture from group 2 ( pretreated with mpeg - nonsense peptide ) and group 3 ( pretreated with unaltered free α125 - 148 ) and an additional group of mice without any treatment prior to the immunizations with achr ( group 5 ). the antibody levels in group 3 were consistently greater than the other two controls . antibodies against peptide α125 - 148 were not detectable in group 4 mice . effect of pretreatment with mpeg -( α125 - 148 ) on the t cell proliferative response the profiles of t - cell responses in eamg - positive ( pretreated with mpeg - nonsense peptide ) and eamg - negative mice which had been treated with mpeg ( α125148 ) were mapped with the synthetic overlapping peptides corresponding to the main extracellular domain ( residues 1 - 210 ) of the a chain of torpedo achr . lymph node cells from each group of mice were pooled separately based on their electrophysiological status for eamg . the t cell proliferation profiles did not show meaningful differences among the groups regardless of their electrophysiological status ( fig1 ). the peptide recognition profile by achr - primed t cells obtained here is in agreement with the profile previously reported for this mouse strain ( yokoi et al ., 1987 ; pachner et al ., 1989 ). autoantibody - mediated mechanisms which have been demonstrated at the motor end plates in both eamg and human myasthenia gravis include 1 ) pharmacological blockade of the acetylcholine binding site ; 2 ) an increased rate of receptor degradation due to cross - linking of adjacent receptors , 3 ) an activation of the complement - mediated membrane lysis and 4 ) an alteration of the ion channel properties of the receptor ( ashizawa and appel 1985 ). previous studies with synthetic α125 - 148 and with overlapping synthetic peptides which comprised the entire extracellular domain of the α subunit of achr have illustrated the pharmacological and immunological ( mulac - jericevic et al ., 1987 ) importance of this region . the sequence of the region α125 - 148 is highly conserved among species . it binds acetylcholine ( mccormick and atassi , 1984 ) and contains a universal binding region for long and short α - neurotoxins ( mulac - jericevic and atassi , 1987b ; ruan et al ., 1990 , 1991 ). because of its direct involvement in the binding of acetylcholine and since the affinity of the antibodies to the receptor is several orders of magnitude higher than that of acetylcholine , the antibodies are capable of effectively blocking the acetylcholine binding site . thus , the inventors reasoned that suppression of the antibody response to this region might alleviate the pharmacological blockade of the acetylcholine binding site , leading to the suppression of the development of eamg . it has been shown ( abuchowski et al ., 1977 ; lee and sehon , 1977 , 1978a ; king et al ., 1977 , 1979 ; davis et al ., 1980 ; sehon , 1989 ) that antibody responses to proteins can be modulated by protein - mpeg conjugates . it was not known , however , whether the approach may be used to obtain epitope - specific suppression of antibody responses to a preselected region of a protein . the results disclosed here demonstrate that injections of mice with mpeg -( α125 - 148 ) suppressed the development of electrophysiological eamg induced by subsequent immunizations with whole torpedo achr , and this was accompanied by a suppression of autoantibody responses restricted to α125 - 148 . these findings suggest that suppression of the antibody responses against this region rescued acetylcholine - binding sites on achr from blockade by such antibodies . other mechanisms may also play a role , however . antibodies against the e subunit may be twice as likely to cross - link the adjacent receptors as antibodies against the other subunits , since the achr is a pentamer consisting of two α subunits and one each of the β , γ and δ subunits . because of this , and the fact that the region α125 - 148 is a major site of recognition by autoantibodies in eamg , it is very likely that the population of antibodies directed against this region plays an important role in the development of eamg through accelerated receptor degradation . likewise , the decrease in this population of autoantibodies may lead to attenuation of the other pathophysiological mechanisms in eamg . the suppression of the electrophysiological eamg was incomplete . since the suppression of the antibody responses to region α125 - 148 was also incomplete , the remaining antibody activities against this region can partially account for the residual disease activity . antibodies directed against other epitopes were not suppressed by the mpeg -( α125 - 148 ) conjugate and may also produce alterations of the synaptic transmission at the motor end plates through accelerated receptor degradation or by allosteric effects on the acetylcholine binding site and would thus have pathogenic activities . pretreatment with the mpeg -( α125 - 148 ) conjugate , followed by immunization with whole achr , caused specific decrease of antibody responses directed against region α125 - 148 , suggesting that the conjugate induces immunosuppression through the regulatory mechanisms involving specific epitope recognition . one of the first steps in immune regulation takes place in the presentation of epitopes to t cells by antigen presenting cells . it has been shown ( holford strevens et al , 1987 ), in mice which had developed tolerance to native ovalbumin via intraperitoneal injections of an ovalbumin - mpeg conjugate , that the presentation of mpeg - modified antigen to t helper ( th ) cells by peritoneal adherent cells was less efficient than the presentation of native antigen . however , since changes in the t - cell responses to α125 - 148 or to other achr α - chain regions were not detected , it is unlikely that the presentation of mpeg -( α125 - 148 ) to th cells is impaired . another mechanism of tolerogenicity caused by the mpeg -( α125 - 148 ) conjugate may involve t suppressor ( ts ) cells . a passive transfer of specific ts cells activated by mpeg - antigen conjugates to syngeneic mice has been shown to cause antigen - specific immunosuppression in the recipient animals , suggesting that induction of antigen - specific ts cells and release of suppressor lymphokines from these cells may play important roles ( lee et al ., 1981 ; mokashi et al ., 1989 ; sehon et al ., 1989 ). further experiments are needed to elucidate the role , if any , of ts cells in the immunosuppression caused by the mpeg - peptide conjugate . immunosuppression of antibody responses mediated by an mpeg - epitope conjugate may also operate at the level of t - b cell collaboration due to impairment of direct contact of the epitope - specific b cells with the conjugate resulting in central tolerance ( sehon and lang , 1986 ). differences might be expected , however , between the presentation and recognition of mpeg - protein conjugates and mpeg - peptide conjugates because of profound differences in their architecture . in conclusion , these data suggest that mpeg - modified peptides corresponding to pathogenic autodeterminants of achr may promise an effective immunospecific treatment for myasthenia gravis in the future . furthermore , the general approach of epitope directed immunosuppression by well - defined tolerogenic mpeg - peptide conjugates offers useful refined strategies for modulation of antibody responses to immunopathogenic sites on multideterminant complex protein antigens . because it provides a means to suppress the production of antibodies against the pathogenic epitopes , its application should not be restricted to autoantigens or alloantigens but should also be applicable to allergens . mpeg itself does not seem to have any harmful or toxic effects in humans . allergens conjugated to mpeg have already been safely administered to human subjects suffering from asthma ( mosbech et al ., 1990 ) and honeybee venom allergy ( muller et al ., 1987 ) with therapeutic effects [ these references as they pertain to methods of treatment of patients with undesirable immune responses , are specifically incorporated by reference herein ]. sehon ( 1988 ) has suggested new clinical applications of mpeg conjugates to arrest the progress of hiv infection to full blown acquired immunodeficiency syndrome ( aids ) in asymptomatic hiv - seropositive individuals . as opposed to the treatment protocols suggested in these prior art approaches which were limited by the masking of the epitopes by the tolerogenic polymer , the present invention exposes the epitope without masking or altering the conformation of a whole native polypeptide antigen ( sehon 1988 ). a method of treatment for an immune disease such as myasthenia gravis , thus , is likely to follow closely these previous drug treatment protocols which used whole antigen . these approaches would be followed except that one would take advantage of the substantial improvement of there being no requirement to take into consideration the masking of the epitopes on the surface of the derivatized whole antigen . thus , as in sehon ( 1988 ) it was anticipated that the antigen binding capacity of antibodies directed against the allergen would be markedly reduced if not totally impaired as a result of conjugation of the allergen with mpeg . for that reason , in previous studies it was necessary to ensure the efficacy of the antibodies directed to the allergen by including a two - phase approach . in the first stage ( immunosuppressive ), a series of injections of tolerogenic mpeg conjugates of the antigen would be made for the induction of the immunosuppression as to the various epitopes represented on the whole antigen . in the second ( effector ) stage , a series of non - conjugated antigens would be injected , either with or without intermittent injections of the tolerogenic derivatives . in the present invention , since there is no masking of the epitope by the tolerogenic polymer , there is no need to follow the injection of mpeg conjugated peptide epitopes with non - conjugated peptides . this represents a substantial improvement over the prior art approaches . these reagents will then be prepared into an immunotherapeutic reagent . immunoassays for disease detection and methods for screening potential reagents for efficacy in treatment of immune diseases the antigenic sites of proteins may either by continuous ( i . e ., occupy continuous segments of the protein chain ) or discontinuous ( i . e , formed by residues that may be distant in the sequence , but come into close spatial proximity in the three - dimensional structure ) ( atassi , 1975 ; atassi , 1978 ; atassi and smith , 1978 ). for a protein of an unknown antigenic structure , the two alternative site architectures should be anticipated . at the present time , any discontinuous antigenic sites , should they exist on achr , cannot be investigated and mimicked without knowledge of the achr three - dimensional structure , which is yet to be determined . in order to localize the continuous regions responsible for autoantibody binding on human achr α - chain , the overlapping synthetic peptide approach was employed ( kazim and atassi , 1980 ; kazim and atassi , 1982 ; mulac - jericevic et al . 1988 ; mulac jericevic and atassi , 1987a ; mulac - jericevic and atassi , 1987b ). this approach has enabled the localization of protein binding sites of diverse activities ( atassi , 1975 ; atassi , 1978 ; atassi , 1984 ; atassi , 1988 ). the human achr α - chain peptides were designed to cover the entire extracellular part ( residues α1 - 210 ) of the subunit . eighteen peptides were made ( fig1 ) that had a near uniform size of 16 or 17 residues and each peptide overlapped its adjacent neighbors by five residues ( mulac - jericevic et al . 1988 ). in addition , a peptide ( α262 - 276 ) corresponding to an extracellular connection between two transmembrane regions ( atassi et al . 1988 ) was investigated . it is proposed that the antigenic peptides or analogs of the same peptides of the invention will find utility as antigens in immunoassays for the detection of anti - peptide antigen - reactive antibodies . turning first to immunoassays , in their most simple and direct sense , certain immunoassays of the invention include the various types of enzyme linked immunosorbent assays ( elisas ) known to the art . however , it will be readily appreciated that utility is not limited to such assays , and useful embodiments include rias and other non - enzyme linked antibody binding assays or procedures . by way of example , in the elisa assay , peptides incorporating the native antigen sequences ( epitopes ) are immobilized onto a selected surface , preferably a surface exhibiting a protein affinity such as the wells of a polystyrene microtiter plate . after washing to remove incompletely adsorbed material , one will desire to bind or coat a nonspecific protein such as bovine serum albumin ( bsa ) or casein onto the well that is known to be antigenically neutral with regard to the test antisera . this allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface . after binding of antigenic material to the well , coating with a non - reactive material to reduce background , and washing to remove unbound material , the immobilizing surface is contacted with the antisera or clinical or biological extract to be tested in a manner conducive to immune complex ( antigen / antibody ) formation . such conditions preferably include diluting the antisera with diluents such as bsa , bovine gamma globulin ( bgg ) and phosphate buffered saline ( pbs )/ tween . these added agents also tend to assist in the reduction of nonspecific background . the layered antisera is then allowed to incubate for from 2 to 4 hours , at temperatures preferably on the order of 25 ° to 27 ° c . following incubation , the antisera - contacted surface is washed so as to remove non - immunocomplexed material . a preferred washing procedure includes washing with a solution such as pbs / tween , or borate buffer . following formation of specific immunocomplexes between the test sample and the bound antigen , and subsequent washing , the occurrence and even amount of immunocomplex formation may be determined by subjecting same to a second antibody having specificity for the first . of course , in that the test sample will typically be of human origin , the second antibody will preferably be an antibody having specificity in general for human ig . to provide a detecting means , the second antibody will preferably have an associated enzyme that will generate a color development upon incubating with an appropriate chromogenic substrate . thus , for example , one will desire to contact and incubate the antisera - bound surface with a urease or peroxidase - conjugated anti - human igg for a period of time and under conditions which favor the development of immunocomplex formation ( e . g ., incubation for 2 hours at room temperature in a pbs - containing solution such as pbs - tween ). after incubation with the second enzyme - tagged antibody , and subsequent to washing to remove unbound material , the amount of label is quantified by incubation with a chromogenic substrate such as urea and bromocresol purple or 2 , 2 &# 39 ;- azido - di -( 3 - ethyl - benzthiazoline - 6 - sulfonic acid [ abts ] and h 2 o 2 , in the case of peroxidase as the enzyme label . quantification is then achieved by measuring the degree of color generation , e . g ., using a visible spectra spectrophotometer . it will , of course , be known to those skilled in the art that the chromatogenic protocol outlined above may be substituted by a radiological procedure such as the use of radioactive isotopes of iodine . an important aspect of the invention is the use of methods of the invention in screening assays for the identification of substances which may immunosuppress or otherwise modify or alter the undesirable immune response . the use of synthetically produced peptides ( epitopes ) is of particular benefit because the naturally occurring antigen may only be present in only small quantities and may difficult to purify from other immunogenic substances . moreover , this allows one a ready source of a wide range of potential epitopes representing various regions on the surface of a polypeptide antigen . the invention also provides access to human epitopes which may be difficult to produce otherwise if one is limited to collection of the native antigen from human tissues . even so , by use of the human - derived epitopes in animal models , the sensitivity to various candidate substances can be first screened prior to human trials . the importance of this is quite significant in that it indicates that where one seeks to identify a compound , e . g ., that may function to immunosuppress the disease in man , that one should employ human version of a particular epitope of a particular antigen for the screening assay . the screening assays of the invention , in preferred embodiments , conveniently employ the animal model most directly mimicking the disease in humans . the battery of tests shown in example ii above for the disease model for myasthenia gravis are illustrative of the types of tests that can be used , e . g ., electophysiological studies , radioimmunoassays , t - cell proliferations assays , etc . in that most such screening assays in accordance with the invention will be designed to identify agents useful in inhibiting the undesirable immune response , preferred assays will typically employ the native antigen from which the peptides are derived in some aspect . thus , it is preferred that a source of the native antigen be available . there are believed to be a wide variety of embodiments which can be employed to determine the effect of a candidate substance such as a tolerogenic epitope - specific peptide on the immune disease of the invention , and the invention is not intended to be limited to any one such method . however , it will generally be desirable to employ a system wherein one can measure the ability of the candidate substance to immunosuppress the disease symptoms in the model . one method employed by the inventors uses a mouse model for the screening of candidate epitopes capable of suppressing the experimental disease symptoms in mice . similar studies have been accomplished using rat models of human allergy responses to ra3 . as mentioned previously , grave &# 39 ; s disease has well - known animal analogs . in preferred assays , the admixture containing the tolerogenic peptide is injected at various intervals into a test subject and allowed to immunosuppress for a selected amount of time , and the resultant animals are tested for reduction of symptoms of the particular immune disease . then , one simply measures the amount of each reduction in symptoms of the disease , e . g ., versus a control to which no candidate substance has been injected . this measurement can be made at various time points where dosage rate data is desired . from this , one may determine the ability of the candidate substance to alter or modify the immune response of the disease . human blood samples were collected in heparinized tubes by venipuncture from patients with a confirmed diagnosis of mg , other neurological or autoimmune diseases and also from normal volunteers . the plasma was separated by centrifugation ( 3 , 000 rpm , 2 ° c ., 10 min ) and stored at 4 ° c . in 0 . 05 % sodium azide . the samples were used within three weeks . fifteen patients with mg were included in this study . the clinical diagnoses were confirmed by the presence of one or more of : ( a ) detectable anti - achr antibody by a classical method ( vincent and newson - davis , 1985 ); ( b ) a positive edrophonium test ; and ( c ) characteristic electrophysiological abnormalities at the motor endplates ( patten , 1978 ) ( table 1 ). table 1 . sup . 1__________________________________________________________________________ edroph - patient mg achr - ab onium thymusnumberage sex class ( nm ) emg test duration mediation histology__________________________________________________________________________ 1 74 m iia 155 . 0 pos pos 5 ( yrs ) pred 10 mg qod no thymectomy 2 67 m iic 84 . 40 pos pos 3 ( yrs ) pyrid 30 mg tid no thymectomy cmp 100 mg pd pred 50 mg qd 3 70 f i 0 . 90 neg pos 7 ( yrs ) none no thymectomy 4 35 f iia 8 . 18 pos pos 19 ( yrs ) pred 50 mg gd hyperplasia 5 34 f iib 6 . 28 neg pos 26 ( yrs ) pyrid 60 mg qid no thymectomy 6 31 f iia 263 . 70 pos pos 9 ( yrs ) pyrid 60 mg bid hyperplasia 7 65 f iia 39 . 75 neg pos 2 ( yrs ) pyrid 120 mg qid thymoma 8 67 m iia 1 . 84 pos pos 18 ( yrs ) pyrid 90 mg q4h no thymectomy 9 17 f iia 71 . 18 pos pos 9 ( yrs ) none hyperplasia10 33 f iib 4 . 30 pos pos 11 ( yrs ) pred 10 mg qod hyperplasia11 61 f iia neg pos pos 8 ( yrs ) pred 15 mg qod histologic12 37 f iia 0 . 40 pos pos 15 ( yrs ) none histologic13 48 f iia 3 . 23 pos pos 1 ( yr ) pyrid 60 mg q4h thymoma14 25 f iia 52 . 00 pos pos 2 ( yrs ) pyrid 60 mg gid hyperplasia15 41 m n / a neg pos pos 8 ( yrs ) pyrid 60 mg qid n / a__________________________________________________________________________ . sup . 1 abbreviations used in table 1 are : mg class : modified osserman &# 39 ; s classification ( patten , 1978 ) achrab : normal range : & lt ; 0 . 2 nm of binding sites for bgtx ( 0 . 2 nm = normal mean value + 3 standard deviations ) medications : pred : prednisone , pyrid : pyridostigmine bromide , cpm : cyclophosphamide all the medications were given orally . emg : electromyography with repetitive nerve stimulation ( patten , 1978 ) n / a : no available fifteen control patients with disorders other than mg were included in the study . the diagnoses of these patients included : autoimmune polyneuropathy ( one ), chronic inflammatory demyelinating polyradiculoneuropathy ( two ), multiple sclerosis ( one ), optic neuritis ( one ), polymyositis ( two ), mononeuropathy multiplex due to vasculitis ( one ), motor axonopathy with anti - gm1 antibody ( two ), amyotrophic lateral sclerosis ( one ), leukoencephalitis ( one ), isaac - merten syndrome ( one ), arachnoiditis ( one ) and spinocerebellar degeneration ( one ). normal subjects included ten males and five females between 22 and 49 years old . eighteen consecutive 16 - or 17 - residue peptides ( fig1 ), overlapping one another by 5 residues and spanning the entire extracellular part ( residues α1 - 210 ) of human achr α - chain ( mulac - jericevic et al . 1988 ), were used to map the continuous autoantigenic ( also often called myasthenogenic ) regions on the extracellular part of the α - chain . in addition , a peptide corresponding to the inter - transmembrane region α262 - 276 , seq . id no . 25 , ( atassi et al . 1988 ) was studied ( fig1 ). the peptides were synthesized , purified and characterized as described ( mulac - jericevic et al . 1988 ; mulac - jericevic and atassi , 1987a ; mulac - jericevic and atassi , 1987b ). another eighteen overlapping peptide spanning the entire extracellular part ( residues α1 - 210 ) of t . californica achr α - chain were synthesized ( mulac - jericevic et al . 1987 ) and used to raise anti - peptide antibodies and to test the coupling efficiency of the synthetic peptide to 96 - well co - bind plates ( covalent binding microtiter plate , micro membranes , inc ., n . j .). anti - peptide antibodies were raised in outbred mice by immunization in the hind footpads of 50 μg of synthetic peptide in 100 μl emulsion prepared from equal volumes of pbs and freund &# 39 ; s complete adjuvant . the immunizations were done on day 1 , day 15 , day 30 and day 45 . rabbit immune igg fractions with specificity against human igg and human igm and mouse igg and igm were obtained from dako co . ( dako - immunoglobulins a / s , denmark ) and accurate chemical scientific corp . ( westbury , n . y . ), respectively . 1 . 0 mg of peptide was dissolved in 5 μl of dimethylformamide and then diluted to 50 μg / ml with 0 . 15m nacl in 0 . 01m sodium phosphate buffer , ph 7 . 2 , containing 0 . 01 % thimerosal ( pbs ). aliquots of the peptide solution , were applied to wells of the 96 - well co - bind plate . the plates were incubated at room temperature overnight with continuous gentle rocking . after washing three times with pbs , the wells were quenched ( 3 hrs ., room temperature ) with 0 . 2 ml of 1 . 0m glycine - naoh , ph 7 . 2 . after washing the wells three times with pbs , an aliquot ( 100 μl ) of a 0 . 1 % solution of casein in pbs were added to each well and the plates were incubated at 37 ° c . for 90 min to block the remaining reactive groups on the wells . the plates were then washed three times with pbs and incubated ( 37 ° c ., 4 hrs ) with 100 μl of plasma or mouse antiserum ( 1 : 200 or 1 : 500 dilution with pbs containing 0 . 1 % casein ). for binding to peptide α182 - 198 , the plates were blocked with , and plasma were diluted in , 0 . 5 % ( instead of 0 . 1 %) casein in pbs . after washing four times with pbs , the plates were incubated ( 4 ° c ., overnight ) with 100 μl of rabbit anti - human ( igg + igm ) or anti - mouse ( igg + igm ) ( 2 . 5 μg / ml ) in pbs containing 0 . 1 % casein . the plates were washed four times with pbs and incubated ( 3 hrs ., room temperature ) with 125 i - labeled protein a ( 2 × 10 5 cpm ) in 100 μl of pbs - 0 . 1 % casein . after washing five times with pbs , bound radioactivity was removed from the wells , by the addition of 100 μl of 5 % sds in 0 . 2n naoh . the plates were allowed to stand for 20 min at room temperature and the solutions were then transferred quantitatively to small test tubes for counting . this procedure was repeated three times , and the solutions in each case were added to the respective tubes . the samples were counted on a beckman 4000 gamma counter . the radioactivity bound by three unrelated proteins ( casein , cytochrome c and bovine serum albumin ) and a nonsense peptide ( essgtgiessgtgi ) ( atassi et al . 1987 ) were used as controls to correct for nonspecific binding . it should be noted that in other experiments , in which the plates were blocked and all dilutions were made with pbs containing human adult hemoglobin or bovine serum albumin , the non - specific binding was considerably higher than when casein was used . to confirm that the synthetic human achr peptides can be coupled to co - bind plate , they were used to raise anti - peptide antibodies in mice . the peptides were bound onto the well of co - bind plates , followed by reaction with the respective anti - peptide antibody , pre - diluted 1 : 500 ( v / v ) with pbs / 0 . 05 % casein . the results ( table 2 ) showed that each peptide was able to bind to its own antibody . the results indicated that achr peptides were in fact coupled to the wells . table 2______________________________________binding of antibodies against human achr α chain peptides tothe peptides on co - bind plate . sup . a ) antibody binding topeptide seq . id immunizing peptidebinding no . ( cpm ) a ) ______________________________________ α1 - 16 7 78940α12 - 27 8 93038α23 - 38 9 71893α34 - 49 10 62360α45 - 60 11 82404α56 - 71 12 36038α67 - 82 13 22884α78 - 93 14 4433 α89 - 104 15 29199α100 - 115 16 57717α111 - 126 17 51177α122 - 138 18 64218α134 - 150 19 52077α146 - 162 20 13649α158 - 174 21 14799α170 - 186 22 71323α182 - 198 23 68217α194 - 210 24 66804______________________________________ . sup . a ) the cpm values have been corrected for nonspecific binding to casein ( 1 . 2 - 5 % of the total binding to the peptides ). the antisera were diluted 1 : 500 ( v / v ) with pbs / 0 . 05 % casein for this assay . the results of the binding profiles of autoantibodies from nine mg patients are summarized in fig1 . the figure also shows the binding profiles of plasma from nine normal controls . the results showed that the autoantibody recognition was directed essentially against four regions broadly localized within ( but may not necessarily include all of ) residues α10 - 30 , α111 - 145 and α175 - 198 and , less frequently , residues α45 - 77 . not all the regions were necessarily recognized by any given mg plasma . for example in mg1 plasma , the autoantibody response to region α182 - 198 , seq . id no . 23 , was the most immunodominant , whereas in mg6 plasma , three of the regions were recognized and in mg7 all four regions were recognized . other examples can be seen in fig1 . in general , the region residing within the consecutive overlapping peptide α170 - 186 ( seq . id no . 22 ) and α182 - 198 ( seq . id no . 23 ) was recognized by autoantibodies of all the individuals examined and was immunodominant in - most cases . of the 19 peptides , peptides α182 - 198 ( seq . id no . 23 ), α122 - 138 ( seq . id no . 18 ), α111 - 126 ( seq . id no . 17 ) and α12 - 27 ( seq . id no . 8 ) were recognized by autoantibodies in 9 , 8 , 6 and 8 out of 9 individuals , respectively . the distribution of the responses to each one of these peptides in nine mg patients in summarized in fig1 and compared to the binding distribution obtained with a similar number of normal , healthy individuals . for each one of these four peptides , the mean binding activity was clearly higher in mg plasma than in normal individuals ( p & lt ; 0 . 001 for α12 - 27 , p & lt ; 0 . 002 for α111 - 126 , p & lt ; 0 , 001 for α122 - 138 and p & lt ; 0 . 001 for α182 - 198 ). however , because of the wide range of the binding distribution , none of these peptides could , by itself , serve as a specific marker for mg diagnosis ( fig1 ). since none of the active peptides could be relied upon for a clear diagnosis of mg , an equimolar mixture of the four most frequently active peptides ( residues α12 - 27 [ seq . id no . 8 ], α111 - 126 [ seq . id no . 17 ], α122 - 138 [ seq . id no . 18 ] and α182 - 198 [ seq . id no . 23 ]) was used to determine autoantibodies in 15 mg plasma samples . the results are summarized in fig1 and are compared with the binding levels obtained with 15 plasma samples from other neurological or autoimmune diseases and samples from normal individuals ( supra ). the 15 mg samples gave considerably higher amounts of antibody binding ( mean net cpm , 5278 ± 1398 ) than the binding obtained with the samples from the other neurological or autoimmune disease ( mean net cpm , 956 ± 652 ; p & lt ;& lt ; 0 . 001 ) and the normal individuals ( mean net cpm , 518 ± 564 ; p & lt ;& lt ; 0 . 001 ). the 15 mg samples clearly clustered at a higher level than the 15 samples from other neurological or autoimmune diseases and the 15 normal individuals , without any overlap with the two control groups . this is due to a complementary effect of the mixture on the binding activities of mg plasma . the mg samples which showed low binding activities for certain peptides had clearly higher binding activities for other peptides ( fig1 ). for example , mg2 had low binding activities for α111 - 126 and α182 - 198 ( overlapping with control values ), but showed clearly higher binding activities for α12 - 27 and α122 - 138 than any control plasma . the situation was similar for all mg plasma except for mgs . with the equimolar mixture of the four peptides , the low binding activities to certain peptides were complemented with the higher binding activities to other peptides in each mg plasma , while binding activities of control plasma were kept low . summary of the continuous antigenic regions recognized by autoantibodies against human achr in mg patients the autoantibody binding profile to the human achr peptides showed that four broad regions were generally recognized . however , except for peptide α182 - 198 , not all the peptides within these four regions were positive with all the antisera . it was shown previously that , in the immune responses to a multideterminant complex protein antigen , the response to each site is under separate ir gene control ( okuda et al . 1979 ). autoimmune responses to autoantigenic sites are also under genetic control ( yokota et al . 1980 ; david and atassi , 1982 ). the results with the mg plasma indicate that the autoimmune responses to the individual antigenic sites are each under genetic control . because of this complication , no universal single peptide was found which will bind with anti - achr autoantibodies in all mg plasma . therefore , four peptides ( α12 - 27 , α111 - 126 , α122 - 138 and α182 - 198 ) were selected because each was able to bind antibodies in the majority of the plasma samples investigated . the cocktail of these peptides enabled the detection of the autoantibodies in all 15 mg plasma samples whereas control plasma from 15 patients with other neurological or autoimmune disorders as well as 15 normal healthy individuals were essentially negative showing that no significant amounts of antibodies against these regions of achr were present in these individuals . using these 18 human achr peptides , the peptide t - cell recognition profiles were determined for autoimmune t - cell lines that were prepared from peripheral blood lymphocytes of several mg patients by passage in vitro with an equimolar mixture of the peptides ( oshima et al . 1990 ). it was found that the profiles of the peptides recognized by the autoimmune t cells were different among the five t - cell lines , consistent with genetic control operating at the level of the individual recognition sites . in the present work , plasma samples were obtained from two of the mg patients ( mg4 and mg6 ) for whom the t - cell recognition profile was determined ( oshima et al . 1990 ). it will , therefore , be valuable to compare , for each of these patients , his / her autoimmune antibody and t - cell recognition profiles . the comparison is summarized schematically in fig1 . the autoimmune t - cell line from mg4 recognized four peptides , only one of which ( peptide α23 - 38 ) was also recognized by autoantibodies . two t - cell recognition regions , α1 - 17 and α89 - 104 , shared an overlap with antibody recognition regions within residues α12 - 27 and α100 - 115 , respectively . it is very likely that when the precise boundaries of the respective autoimmune t - and b - cell recognition sites are determined , the overlaps between these recognition features will be more extensive ( atassi et al . 1987 ). it should be noted that the overlapping peptide strategy ( kazim and atassi , 1980 ; kazim and atassi , 1982 ) is not expected to give the boundaries of the autorecognition sites but rather to localize the maximal continuous regions within which these sites would reside . at any rate , one t - cell auto - determinant within region α146 - 162 ( seq . id no . 20 ) was exclusively a t - cell recognition feature , since no autoantibodies were directed to it in this individual . the t - cell line from mg6 recognized three auto - determinants , two of which ( α111 - 126 and α182 - 198 , seq . id nos . 17 and 23 , respectively ) coincided completely with regions recognized by autoantibodies while the third ( α146 - 162 ) shared an overlap with the autoantibody binding region within residues α134 - 150 ( seq . id no . 19 ). it is evident , therefore , that in given individuals , autoantibodies and autoimmune t - cells may recognize similar sites , but there are in addition sites that are recognized only by antibodies and sites that are recognized only by t cells . this is consistent with previous findings on t - and b - cell recognition of proteins in foreign hosts ( for review , see ref . ( atassi , 1984 ) and in human allorecognition of class ii mhc molecules ( lindstrom et al . 1976 ). the results reported here and previously with autoimmune t - cells suggest that the pathogenesis of this autoimmune disease is variable at the molecular - cellular level . by using the overlapping peptide strategy employed here , the present inventors have mapped the regions of a - neurotoxin ( both bgtx and cobratoxin ) binding on the extracellular part of the α - subunits of torpedo californica ( mulac - jericevic and atassi , 1987a ; mulac - jericevic and atassi , 1987b ) and human ( mulac - jericevic et al . 1988 ) achr . a major toxin binding region was found ( mulac - jericevic et al . 1988 ; mulac - jericevic and atassi , 1987a ; mulac - jericevic and atassi , 1987b ) to reside within the peptide α122 - 138 and two minor regions occur within peptides α34 - 49 and α194 - 210 . the results reported here show that peptide α122 - 138 also contains an autoantigenic region in most mg sera . the binding activity of this region with autoantibody and with α - neurotoxin would explain the false negatives obtained with the method that relies on the precipitation by the autoantisera of the 125i - labeled bgtx - achr complex . the region α122 - 138 carries contact residues of the acetylcholine binding sites ( mccormick and atassi , 1984 ). the present finding that autoantibodies also bind to this region provides a molecular explanation for the dysfunction of achr in mg . the present work , in addition to localizing the continuous regions of autoantibody recognition from which the molecular basis for the dysfunction in mg may be better understood , identifies a cocktail of peptides that may be employed successfully to reduce an ambiguous diagnosis . thus , utilizing the materials and methods of the present the present inventors have localized the major continuous auto - antigenic regions on the extracellular part of the α - chain of human achr that are recognized by autoantibodies in mg patients . one of these regions contained contact residues of the acetylcholine binding site . this has provided a molecular explanation for the dysfunction of achr in the autoimmune disease . furthermore , the results permitted comparison of the autoantibody recognition profile with autoimmune t - cell recognition regions on human achr in the same donor . by using an equimolar mixture of the most frequently active peptides , it has been possible to detect the autoantibodies in all the mg sera examined . this peptide mixture provides a molecular basis for reliable diagnosis of the disease . the following references to the extent that they provide procedural details supplementary to those set forth herein , are specifically incorporated herein by reference . abuchowski , a ., van es ., t ., palczuk , n . c . and davis , f . f ., j . biol . chem . 252 : 3578 ( 1977 ). ashizawa , t . and appel , s . h ., springer sem immunopathol . 8 : 177 - 196 ( 1985 ). atassi , m . 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in immunobiology of proteins and peptides v - vaccines : mechanisms , design , and applications , ed . m . z . atassi , pp . 341 , plenum press , new york ( 1988 ). sehon , a . h . and lang , g . m ., in mediators of immune regulation and immunotherapy ( s . k . singhal and t . l . delovitch , eds .) pp . 190 - 203 , elsevier , new york ( 1986 ) sobel , a ., weber , m . and changeux , j . p ., eur . j . biochem . 80 : 215 - 224 ( 1977 ). vincent , a . and newson - davis , j ., j . neurol . neurosurg . and psych . 48 : 1246 - 1252 ( 1985 ). wei , s . i ., wei , c . w ., lee , w . y ., filion , l . g ., sehon , a . h . and akerblom , e ., int . arch . allergy appl . immunol . 64 : 84 - 99 ( 1981 ). yokoi , t ., mulac - jericevic , b ., kurisaki , j . t . and atassi , m . z ., europ . j . immunol . 17 : 1697 - 1702 ( 1987 ). yokota , s ., davis , c . s . and atassi , m . z ., mol . immunol . 17 : 1079 - 1082 ( 1980 ). the present invention has been described in terms of particular embodiments found or proposed to comprise preferred modes for the practice of the invention . it will be appreciated by those of skill in the art that , in light of the present disclosure , numerous modifications and changes can be made in the particular embodiments exemplified without departing from the intended scope of the invention . for example , it will be understood that one may synthesize the peptides of the invention or one may obtain peptide fragments which are either wholly or partially a fragment of a native antigen or a recombinant derivative of such . additionally , while the amino acid sequence selected for a given peptide will typically occur as such in the native antigen , it will be understood that one may choose to substitute similar hydropathic amino acids and that the peptide may contain non - linear portions ( i . e ., such a peptide may represent a discontinuous epitope ) of a given antigen , alloantigen or allergen . similarly , one may wish to add amino acid residues to either one or both termini of the epitope proper which amino acid residues are not relevant to the specificity of the epitope but otherwise facilitate its use or ease of purification , for instance . it is also understood that the tolerogenic polymer molecule may be coupled either to the n - terminal α - nh 2 group or to the c - terminal carboxyl group or , in certain cases , to both termini . all such modifications are intended to be included within the scope of the appended claims . __________________________________________________________________________sequence listing ( 1 ) general information :( iii ) number of sequences : 25 ( 2 ) information for seq id no : 1 :( i ) sequence characteristics :( a ) length : 15 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 1 : glylysvaltyrleuvalglyglyprogluleuglyglytrplys151015 ( 2 ) information for seq id no : 2 :( i ) sequence characteristics :( a ) length : 15 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 2 : gluvaltrparggluglualatrphisalacysaspilelysasp151015 ( 2 ) information for seq id no : 3 :( i ) sequence characteristics :( a ) length : 15 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 3 : proglyglyproaspargphethrleuleuthrproglyserhis151015 ( 2 ) information for seq id no : 4 :( i ) sequence characteristics :( a ) length : 15 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 4 : thrproglyserhispheilecysthrlysaspglnlyspheval151015 ( 2 ) information for seq id no : 5 :( i ) sequence characteristics :( a ) length : 24 amino acids ( b ) type : amino acid ( d ) topology : circular ( ii ) molecule type : peptide ( ix ) feature :( a ) name / key : disulfide - bond ( b ) location : 4 .. 18 ( c ) other information : / note =&# 34 ; internal disulfidelinkages creating peptide loop &# 34 ;( xi ) sequence description : seq id no : 5 : lyssertyrcysgluileilevalthrhisphepropheaspglngln151015asncysthrmetlysleuglyile20 ( 2 ) information for seq id no : 6 :( i ) sequence characteristics :( a ) length : 15 amino acids ( b ) type : amino acid ( d ) topology : circular ( ii ) molecule type : peptide ( ix ) feature :( a ) name / key : disulfide - bond ( b ) location : 4 .. 15 ( c ) other information : / note =&# 34 ; internal disulfidelinkages creating peptide loop &# 34 ;( xi ) sequence description : seq id no : 6 : lysserprocysalatyrlysgluprogluthrthrvalalacys151015 ( 2 ) information for seq id no : 7 :( i ) sequence characteristics :( a ) length : 16 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 7 : sergluhisgluthrargleuvalalalysleuphelysasptyrser151015 ( 2 ) information for seq id no : 8 :( i ) sequence characteristics :( a ) length : 16 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 8 : phelysasptyrserservalvalargprovalgluasphisarggln151015 ( 2 ) information for seq id no : 9 :( i ) sequence characteristics :( a ) length : 16 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 9 : gluasphisargglnvalvalgluvalthrvalglyleuglnleuile151015 ( 2 ) information for seq id no : 10 :( i ) sequence characteristics :( a ) length : 16 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 10 : glyleuglnleuileglnleuileasnvalaspgluvalasnglnile151015 ( 2 ) information for seq id no : 11 :( i ) sequence characteristics :( a ) length : 16 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 11 : gluvalasnglnilevalthrthrasnvalargleulysglnglntrp151015 ( 2 ) information for seq id no : 12 :( i ) sequence characteristics :( a ) length : 16 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 12 : leulysglnglntrpvalasptyrasnleulystrpasnproaspasp151015 ( 2 ) information for seq id no : 13 :( i ) sequence characteristics :( a ) length : 16 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 13 : trpasnproaspasptyrglyglyvallyslysilehisileproser151015 ( 2 ) information for seq id no : 14 :( i ) sequence characteristics :( a ) length : 16 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 14 : ilehisileproserglulysiletrpargproaspleuvalleutyr151015 ( 2 ) information for seq id no : 15 :( i ) sequence characteristics :( a ) length : 16 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 15 : aspleuvalleutyrasnasnalaaspglyaspphealailevallys151015 ( 2 ) information for seq id no : 16 :( i ) sequence characteristics :( a ) length : 16 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 16 : phealailevallysphethrlysvalleuleuglntyrthrglyhis151015 ( 2 ) information for seq id no : 17 :( i ) sequence characteristics :( a ) length : 16 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 17 : glntyrthrglyhisilethrtrpthrproproalailephelysser151015 ( 2 ) information for seq id no : 18 :( i ) sequence characteristics :( a ) length : 17 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 18 : alailephelyssertyrglygluileilevalthrhisphepropheasp151015 ( 2 ) information for seq id no : 19 :( i ) sequence characteristics :( a ) length : 17 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 19 : hisphepropheaspgluglnasnglysermetlysleuglythrtrpthr151015 ( 2 ) information for seq id no : 20 :( i ) sequence characteristics :( a ) length : 17 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 20 : leuglythrtrpthrtyraspglyservalvalalaileasnprogluser151015 ( 2 ) information for seq id no : 21 :( i ) sequence characteristics :( a ) length : 17 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 21 : ileasnprogluseraspglnproaspleuserasnphemetglusergly151015 ( 2 ) information for seq id no : 22 :( i ) sequence characteristics :( a ) length : 17 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 22 : phemetgluserglyglutrpvalilelysgluserargglytrplyshis151015 ( 2 ) information for seq id no : 23 :( i ) sequence characteristics :( a ) length : 17 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 23 : argglytrplyshisservalthrtyrserglyglyproaspthrprotyr151015 ( 2 ) information for seq id no : 24 :( i ) sequence characteristics :( a ) length : 17 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 24 : proaspthrprotyrleuaspilethrtyrhisphevalmetglnargleu151015 ( 2 ) information for seq id no : 25 :( i ) sequence characteristics :( a ) length : 15 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 25 : gluleuileproserthrserseralavalproleuileglylys151015__________________________________________________________________________