MATERIALS AND METHODS .sx ( Ca 2+ -Mg 2+ )-ATPase was purified from rabbit skeletal muscle SR as described previously ( Colyer et al .sx , 1989 ) and gave a preparation which , on polyacrylamide gels stained with Coomassie Blue , was >97% pure ( Gould et al .sx , 1987) .sx mAbs were prepared as described in Colyer et al .sx ( 1989 ) and were purified from ascites fluid by precipitation with 40%-satd .sx formula followed by dialysis against 2x1 litre of phosphate-buffered saline ( PBS ; 137 m M-NaCl , 2.7 m M-KCl , 8.1 m M- formula , 1.5 m M- formula , pH 7.2 ) , giving a preparation containing approx. 50% mAb by weight at a protein concentration of 1-5 mg/ml .sx Synthesis and use of hexapeptides .sx Hexapeptides were synthesized on blocks of polyethylene pins designed to fit into 96-well microtitre plates , using kits supplied by Cambridge Research Biochemicals , as described by Geysen et al .sx ( 1987 ) by stepwise chain elongation using Fmoc ( fluorenylmethoxycarbonylamine ) chemistry ( Van Regenmortel et al .sx 1988) .sx NN'-Dimethylformamide ( DMF ; Romil Chemicals Ltd. ) was stored over molecular sieve 4A ( BDH ) until assay for free amino groups using fluorodinitrobenzene showed it to be free of dimethylamine .sx All other reagents were AnalaR grade , and water was distilled .sx The pre-derivatized pins were supplied with Fmoc-alanine linked via hexamethylenediamine to the pins .sx The Fmoc N-terminal protecting group was removed from the pins and from subsequent coupled Fmoc-amino acids by washing with 20 % ( v/v ) piperidine in DMF for 30 min at room temperature with shaking .sx The pins were then washed for 5 min with DMF , four times with methanol for 2 min , air-dried for 10 min and washed for a further 5 min with DMF .sx Fmoc-amino acid active esters ( Fields & Noble , 1990 ) were dissolved in DMF containing 1-hydroxybenzotriazole and the mixtures were dispensed into the appropriate wells of a polypropylene e.l.i.s.a. plate according to a computer-generated synthesis schedule .sx The pins were inserted into the wells and coupling was allowed to proceed at room temperature for at least 18 h. The blocks were then removed and washed as above .sx After synthesis of the hexapeptides and de-protection of the N-terminal amino acid , the N-terminal amino acid was acetylated by placing the block of pins into a mixture of DMF , acetic anhydride and di-iso - propylethylamine ( 50 :sx 5:1 , by vol. ) and incubating for 90 min at room temperature .sx The blocks were then washed as above , Side - chain-protecting groups were removed by placing the block of pins in a mixture containing trifluoroacetic acid , phenol and ethanedithiol ( 38 :sx 1:1 , v/w/v ) for 4 h at room temperature .sx The pins were then washed twice with dichloromethane for 2 min , twice with 5% di-isopropylethylamine in dichloromethane for 5 min , and once with dichloromethane for 5 min before air - drying and finally washing in water for 2 min and methanol for 18 h before drying in vacuo .sx Enzyme immunoassays were carried out by first precoating the pins for 1 h by insertion into wells of e.l.i.s.a. plates containing buffer A ( 1% ovalbumin , 1% BSA , 0.1% Tween 20 and 0.05% NaN 3 in PBS) .sx Pins were then incubated overnight at 4 degree C in e.l.i.s.a. plates containing mAb at a 1 :sx 500 dilution with the buffer mixture described above .sx The pins were then washed three times for 10 min with PBS containing 0.05% Tween 20 , with shaking .sx Bound antibody was detected by incubation for 1 h at 25 degree C in e.l.i.s.a. plates containing second antibody conjugated to horseradish peroxidase ( HRP ; Sera-Tec , diluted 1 :sx 1000 in buffer A but in the absence NaN 3 ) .sx The pins were washed as before and then incubated in substrate solution [50 mg of azino-di-3-ethylbenzthiazodinosulphate , 35 mu l of 100 volume H 2 O 2 in 100 ml of citrate/phosphate buffer ( 80m M ) , pH 4.0] in the wells of an e.l.i.s.a. plate .sx The colour was allowed to develop in the dark for 30 min and then the plates were read at 410 nm on a Dynatech MR588 Microelisa Auto Reader .sx To allow re-use , antibodies were removed from the pins by cleaning in a sonication bath with 1% SDS/0 .sx 1% 2-mercaptoethanol/0 .sx 1 M- formula , pH 7.2 at 60 degree C for 30 min , followed by washing in hot distilled water ( 55-60 degree C) .sx Pins were then immersed in boiling methanol for 2 min and dried in air .sx Preparation of polyclonal antibodies .sx Polyclonal antibodies to the purified ATPase were raised in sheep by intramuscular injections of 0.5 mg of ATPase in Freund's complete adjuvant followed by boosts in Freund's incomplete adjuvant .sx Antibodies were purified from antisera by precipitation with 40% formula followed by dialysis against 2x1 litre of PBS .sx Antibodies were immunoprecipitated as follows .sx Samples ( 0.2 ml , containing 0.2 mg of antibody ) were diluted to 10 ml with PBS .sx Purified ATPase ( 1 mg in 100 mu l ) was then added to give a ratio of ATPase/antibody of 5 :sx 1 ( w/w ) , and the sample was incubated for 1 h at room temperature with shaking to allow precipitation of the antibody-antigen complex .sx Samples were then spun at 37000 g for 30 min at 20 degree C , and the supernatant was removed and assayed as described above for the mAbs .sx Preparation of peptides and anti-peptide antibodies .sx Peptides are named according to the residues to which they correspond in the ATPase .sx Peptides were synthesized by the method of Merrifield ( 1986 ) and checked for purity by h.p.l.c. on a reversed-phase C8 column .sx Peptides were coupled to the carrier protein keyhole limpet haemocyanin ( KLH ) before immunization according to the method of Green et al .sx ( 1982) .sx Primary immunizations were carried out with approx. 0.5 mg of peptide-KLH in Freund's complete adjuvant which was injected by the intramuscular route in New Zealand white rabbits on day 1 .sx A booster injection of 0.25 mg of peptide-KLH was given in Freund's incomplete adjuvant by the same route after 28 days , and blood was taken for the production of antisera 7-14 days later .sx Antisera were stored at -70 degree C. Antibody was purified from antisera by precipitation with 40% formula , as described above .sx Further purification was carried out using an ATPase affinity column made by reacting SR vesicles with CNBr-activated Sepharose .sx Antiserum was passed through the column at pH 8.0 and bound antibody was eluted with glycine/HCl ( 0.1 M , pH ) .sx The eluent was immediately neutralized with a few drops of Tris/HCl ( 2 M , pH 7.4 ) and dialysed against 2x1 litre of PBS .sx To study effects of the affinity-purified anti-peptide antibody on the activity of the ATPase , the enzyme was incubated for 30 min with antibody at a 3 :sx 1 molar ratio of antibody to ATPase .sx ATPase activity was determined by a coupled enzyme assay ( Colyer et al .sx 1989) .sx All anti-peptide antibodies were mapped against the sets of pins as described above for the mAbs , and all showed recognition of the appropriate hexameric peptides , with no significant binding to other hexameric peptides .sx figure&caption .sx Competitive e.l.i.s.a. A modified e.l.i.s.a. technique was used to demonstrate that the monoclonal and polyclonal antibodies bound to native ATPase ( Colyer et al .sx , 1989) .sx Antibody ( 0.7 mu g ) was incubated overnight at 4 degree C in PBS/Tween in uncoated e.l.i.s.a. plates with the ATPase ( 2-160 mu g/ml ) either in the native form or after heat denaturation ( 100 degree C for 3 min) .sx Samples were then transferred to e.l.i.s.a. plates coated with the ATPase ( 1 mu g/well ) and e.l.i.s.a. was performed using second antibody conjugated to HRP ( Colyer et al .sx , 1989) .sx RESULTS Competitive e.l.i.s.a. was used to show that mAbs bound to the native ATPase ( Fig. 1) .sx Native and heat-denatured ATPases were incubated overnight with mAb , and the incubations were transferred to e.l.i.s.a. plates coated with native ATPase to determine the amount of free mAb remaining in the incubations .sx Fig. 1 shows that although mAbs Y/1F4 and Y/2E9 bound more strongly to denatured than to native ATPase , the difference was rather small .sx For example , for Y/1F4 , twice as much of the native ATPase than of the denatured ATPase was required to lower the e.l.i.s.a. reading by 50 % .sx Similar results were obtained with the other mAbs ( results not shown ) , confirming that the epitopes for these mAbs are surface-exposed in the native ATPase .sx To define the exact epitopes for the mAbs , a set of overlapping hexameric peptides was synthesized on polyethylene pins arranged to fit the wells of standard microtitre plates ( Geysen et al .sx , 1987) .sx On the basis of our preliminary mapping of the epitopes ( Colyer et al .sx , 1989 ) , we chose to synthesize hexapeptides corresponding to amino acid regions 1-208 , 277-381 and 486-751 .sx Our previous studies suggested that the epitope for mAb Y/1F4 lay between amino acids 500 and 615 ( Colyer et al. .sx , 1989 ; Tunwell et al .sx , 1991) .sx The results of the binding analysis for mAb Y/1F4 with the set of hexapeptides from amino acids 486-751 are shown in Fig. 2( a) .sx mAb Y/1F4 showed strong recognition of hexapeptide NKMFVK , corresponding to residues 510-515 , indicating that this is the epitope for the antibody .sx The adjacent peptides VGNKMF , KMFVKG and MFVGAP gave lower signals , suggesting that these peptides contain only part of the epitope , with the sequence MF ( Met-Phe ) being the most critical part of the epitope .sx As shown in Fig. 2( d ) , mAb Y/2E9 gave a strong signal for hexapeptides LPLAEQ and PLAEQR , suggesting that the epitope was PLAEQ , corresponding to amino acids 662-666 ; the very weak binding to the neighbouring hexapeptides suggest that in this case the sequence PL ( Pro-Leu ) is critical for binding .sx mAb A/4H3 showed binding very similar to that of mAb Y/2E9 , and mAb B/4H3 showed strong binding to peptides PLAEQR and LAEQRE , suggesting that their epitopes are the same as that of mAb Y/2E9 ( Table 1) .sx As shown in Fig. 2( c ) , mAb 1/2H7 bound strongly to hexapeptides DDSSRF , DSSRFM , SSRFME and SRFMEY , so that the major component of its epitope is probably SRF ( Ser-Arg-Phe ) , corresponding to residues 583-585 .sx Also , as shown in Fig. 2( b ) , the epitope for mAb 1/3D2 is SSRFME , corresponding to amino acids 583-588 .sx From our previous epitope mapping experiments ( Colyer et al .sx , 1989 ; Tunwell et al .sx , 1991 ) , epitopes from mAbs B/3D6 , Y/1G4 , Y/1G6 , Y/2D8 , Y/2D11 and Y/3H2 were all thought to lie between amino acid residues 486 and 750 , but for none of these mAbs was any clear pattern of binding to the hexameric peptides observed ( results not shown ) , so that the epitopes for these mAbs cannot be defined using this approach .sx Epitopes for mAbs Y/3H5 , Y/1H12 , Y/2A2 and Y/4D6 have all been suggested to lie between residues 320 and 376 ( Colyer et al .sx , 1989 , Tunwell et al .sx , 1991) .sx Binding of these mAbs was therefore tested with a set of hexapeptides corresponding to amino acids 277-381 .sx The results for mAb Y/4D6 are shown in Fig. 3 and are considerably less clear cut than those described above .sx Binding to hexapeptides .sx AAIPEG , AIPEGL and IPEGLP was strong , suggesting an epitope whose main binding IPEGLP was strong , suggesting an epitope whose main binding determinant is IPE ( Ile-Pro-Glu ) , corresponding to amino acids 307-309 .sx However , equally strong binding was seen to hexapeptide VRSLPS ( residues 333-338 ) which could suggest that the epitope for the mAb is an assembled one , containing both of these regions .sx Slightly weaker binding is also observed to hexapeptides PVHGGS ( residues 282-287 ) and VHGGSW ( residues 283-288 ) ; since hydropathy plots suggest that either residues 288-307 ( MacLennan et al .sx , 1985 ) or residues 295-315 ( Green , 1989 ) make up a transmembrane alpha -helix , these could not be part of the same epitope as residues 307-309 and 333-338 , and binding to these two hexapeptides would then have to be artifactual .sx Alternatively , one set of reactive peptides might be mimotopes of the true epitope .sx The pattern of binding observed with mAb Y/3H5 was very similar to that shown in Fig. 3 for mAb Y/4D6 .sx mAbs Y/1H12 and Y/2A2 only gave weak binding to any of the hexapeptides , and so could not be clearly mapped .sx graphs&captions .sx To check the epitope assignments made above , the peptides shown in Table 1 were synthesized using the method of Merrifield ( 1986 ) , with all peptides containing an N-terminal Cys .sx E.l.i.s.a. was performed using peptide coated on to e.l.i.s.a. plates , and , as shown in Table 1 , Y/1F4 , Y/2E9 and 1/2H7 all bound to their respective peptides .sx No binding of mAb Y/3H5 was observed in e.l.i.s.a. to peptides AVAAIPEGLPAV ( residues 303-314 ) or RRMAKKNAIVRSLPSV ( 324-339 ) , which contain the two possible contributory epitopes for this mAb ( see Table 1 ) ; a negative result in this experiment does not , however , prove that the epitope for the mAb is not contained in the peptides and could , for example , simply indicate that the peptides are coated on to the plastic in conformations unsuitable for binding .sx