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Glycoprotein methods protocols - biotechnology
Synthetic Peptides for Antimucin Antibodies 129129From:Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The MucinsEdited by: A. Corfield © Humana Press Inc., Totowa, NJ12Synthetic Peptides for the Analysis and Preparationof Antimucin AntibodiesAndrea Murray, Deirdre A. O’Sullivan, and Michael R. Price1. IntroductionSince the mid-1980s, the family of high molecular weight glycoproteins known asmucins have evoked considerable interest among those in the field of cancer research.Mucins, which are constituents of mucus, have a lubricating and protective function innormal epithelial tissue (1). However, expression of mucin by the cancer cell is oftenhighly disorganized and upregulated, sometimes to the extent that mucin can bedetected in the circulation of the cancer patient. These changes in expression of mucinobserved in neoplasia have led to the exploitation of some members of the mucinfamily as circulating tumor markers (2,3) or targets for diagnostic imaging (4–6) andtherapy of cancer.The first mucin to have its primary amino acid sequence determined, MUC1, is alsothe most extensively studied. This molecule is highly immunogenic, and a consider-able number of anti-MUC1 monoclonal antibodies (mAbs) and fragments have beenproduced by various methods. Some of these have found applications for radio-immunoscintigraphy and targeted therapy of cancer, and others have been used todetect circulating MUC1. Although such studies have yielded promising results, theirpresent application is somewhat restricted. In this age of genetic and protein engineer-ing, we have, at our disposal, the technology to design antibodies with ideal character-istics of size, affinity, and specificity for any desired application. However, beforeconsidering such ambitions, we must first gain an understanding of the molecularinteractions between epitope and paratope when an antibody binds to its antigen. It isessential that key residues involved in the interaction are identified so that a model ofhow the interaction takes place on a three-dimensional level can be constructed. Thisidentification will enhance our ability to design antibodies with the correct character-istics for our chosen application. 130 Murray et al.1.1. ImmunoassaysBoth enzyme-linked immunosorbant assays (ELISAs) and radioimmunoassays havebeen used in various formats to test antibody binding to synthetic peptides. The indi-rect ELISA has the advantages of being easy to perform, having no requirement forradioactive tracers, and producing results that are simple to interpret. The disadvan-tage of the indirect ELISA is that the procedure requires that the antigen, in this case asynthetic peptide, be immobilized on to the surface of a microtiter plate well. Classi-cally this would be achieved by dispensing a solution of antigen into the wells of amicrotiter plate to allow adsorption, leaving the plate coated with antigen. However,short synthetic peptides adsorbed on to plates in this way provide unpredictable andinconsistent results. This problem may be owing to the fact that the orientation of thepeptide on the plate cannot be controlled or simply that short peptides do not adherewell to polystyrene plates. Several methods of peptide modification have been utilizedto overcome these problems. One such procedure involves preparing branched-chainpolypeptides in which MUC1 immunodominant peptides ware conjugated to a polyl-ysine backbone (7). These polylysine conjugates provide very potent MUC1-relatedantigens for the interrogation of antibody specificity; however, the methodology fortheir preparation is beyond the scope of this chapter. By far the most widely usedmethod for modifying short peptides so that they can be used as antigens in indirectELISA procedures is to conjugate the peptides to a large carrier protein such as bovineserum albumin (BSA) (see Subheading 3.1. and Notes 1–3).1.2. Tethered Peptide Libraries for Exploring Antibody SpecificityThe peptide synthesis techniques developed by Geysen and colleagues (8) repre-sent a significant development in the study of epitopes defined by antibodies reactivewith antigens of known primary structure. Unlike most other methods of simultaneouspeptide synthesis, this technique allows the concurrent synthesis of hundreds to thou-sands of peptides so that libraries can be produced and simultaneously used as targetsfor antibody binding. The peptides are synthesized on derivatized polyethylene orpolypropylene gears that are held on stems (Fig. 1) arranged in a microtitre plate for-mat so that a simple ELISA procedure can be used to measure antibody binding. Pep-tides are tethered via the carboxyl terminus.Several different strategies have been described for peptide sequence design thatall provide different information on epitope structure and the fine specificity of anantibody-peptide interaction. The Pepscan approach has been the most widely usedand involves the synthesis of a set of overlapping peptides that span the length of theantigenic sequence (Subheading 3.2.). In a short peptide sequence, such as that of theMUC1 variable number of tandem repeat (VNTR), each peptide may overlap the nextby all but one amino acid, giving rise to a set of 21 heptapeptides that spans the VNTRsequence (Fig. 2). For larger proteins, it is more appropriate to produce longersequences that overlap each other by less residues, thereby spanning the length of theantigenic sequence with a feasible number of peptides (see Note 4). In the Pepscanapproach, peptides are assayed for antibody-binding capacity by ELISA (Subheading3.3.), and residues that are common to all the antibody-binding pins represent the mini- Synthetic Peptides for Antimucin Antibodies 131Fig. 1. The Multipin Peptide Synthesis System contains detachable polyethylene gears thatfit on to the end of stems. The stems are held in a block in an 8 x 12 microtiter plate format. Thesurface of the gear is derivatized to give a solvent-compatible polymer matrix on which thepeptides are coupled during synthesis. The matrix also provides a two amino acid spacer group.Fig. 2. Schematic representation of the overlapping peptides corresponding to the MUC1VNTR sequence synthesized according to the Pepscan approach to epitope mapping. Antibod-ies are allowed to react with each peptide, and those containing the epitope or minimum bind-ing unit produce positive results. In this example, the epitope can be deduced as consisting ofthe amino acids that are common to all positive pins (7–10). Hence, the epitope is PDTR. 132 Murray et al.mum binding unit or epitope for that antibody (Fig. 2). Having identified the epitopedefined by an antibody using Pepscan, it may be useful to prepare a number of analogsof that sequence in order to investigate the role of individual amino acids in the epitopeand to identify critical contact residues. Such peptide design stategies includeommission analysis, alanine substitution and replacement net (RNET) analysis (seeNotes 5–9).Libraries of peptides on pins can be obtained that comprise 400 different dipeptidesprepared with all possible combinations of the 20 natural amino acids. This approachprovides qualitative information on antibody specificity and permits identification ofsignificant features of an epitope that may contribute to antibody recognition and bind-ing (see Notes 10 and 11).1.3. Purification of Antibodies Using Peptide Affinity ChromatographyThe identification of a linear peptide epitope within a protein sequence facilitatesthe design of peptide affinity matrices that can be used to purify antibodies from bio-logical feedstocks. Such an epitope affinity matrix has been produced by covalentlylinking a synthetic peptide corresponding to the MUC1-immunodominant domain tocyanogen bromide-activated Sepharose (Pharmacia, Uppsala, Sweden) (9). Theresulting matrix was remarkably efficient for the purification of a range of anti-MUC1mAbs from biological feedstocks containing high levels of contaminating proteinssuch as ascitic fluid and hybridoma supernatant (see Note 12).Epitope affinity chromatography matrices have an advantage over other affinityadsorbents in that the antibody is bound to the matrix specifically via the paratope.Thus, eluted antibody is fully immunoreactive and of only the desired specificity.Sepharose-peptide conjugates are simple to prepare and affinity chromatography ismore robust than other conventional chromatographic techniques in terms of columnpacking and operation (see Subheading 3.4., Notes 13–15, and Fig. 3).1.4. General CommentsThe techniques described for the analysis of antimucin antibodies using syntheticpeptides can provide a great deal of information on epitope topography and structure.The identification of critical binding residues within an epitope can provide clues tothe forces and residues involved in the antibody-antigen interaction. However, bear inmind that the use of linear synthetic peptides can only provide a one-dimensionalsolution to what is essentially a three-dimensional problem. Further structural studiessuch as X-ray crystallography, nuclear magnetic resonance spectroscopy, and compu-tational molecular modeling are essential if the knowledge gained is to be confirmedand translated into a useful model on which to base antibody design strategies.The structural information provided by studies such as those previously describedmay be of use in peptide vaccine design. However, the analyses performed so far havebeen mainly concerned with the interaction of murine antibodies, and it may be naiveto assume that the human immune system will process mucin-related antigens in thesame way. Preliminary epitope-mapping studies on human serum would suggest thatthe immune response to MUC1 may differ considerably from that observed in themouse (10). Synthetic Peptides for Antimucin Antibodies 133Finally, it may be owing to the very nature of the mucins that such a wealth of informa-tion has been provided by the techniques described. The VNTR provides a convenientshort sequence on which to base peptide synthesis strategies. In addition, most murineantimucin antibodies analyzed to date have been shown to define short linear determi-nants. It is unlikely that all other proteins and antibodies will be so accommodating.Fig. 3. Schematic representation of the apparatus and reagents needed for the purification ofantibodies by peptide epitope affinity chromatography. 134 Murray et al.2. Materials2.1. Preparation of BSA-Peptide Conjugates1. Conjugation buffer: sodium hydrogen carbonate buffer (0.1 M, pH 8.4).2. BSA: crystalline, greater than 96% pure.3. Glutaraldehyde: when used as a crosslinker must be freshly distilled or high commercialgrade (Sigma, Poole, UK).4. Dialysis buffer: sodium chloride 1% (w/v).2.2. Solid-Phase Peptide Synthesis on PinsAll reagents used in solid-phase peptide synthesis should be of the highest avail-able purity (analytical reagent grade or better) unless stated otherwise.1. Mulitpin Peptide Synthesis Kit (Chiron Mimotopes, Clayton, Victoria, Australia).2. Amino acids: All amino acids recommended for use with the Multipin Peptide SynthesisKit have their α-amino group protected with the 9-fluorenylmethyloxycarbonyl (Fmoc)group. Table 1 appropriate side chain protecting groups. Alternatively, protected aminoacid esters may be used. These have the advantage of requiring no prior activation. How-ever, they are prone to decomposition with prolonged storage and are best stored at –20°C.3. Activators: The activation of protected amino acids with diisopropylcarbodiimide (DIC)/1-hydroxybenzotriazole (HOBt) is recommended, but other coupling reagents can be used.4. N,N-Dimethylformamide (DMF): DMF used in peptide coupling procedures must be pureand free from amines. Several methods may be used to purify DMF (see Note 16).5. Piperidine 20% v/v: used for Fmoc deprotection. Piperidine should be redistilled beforeuse and made up to a 20% (v/v) solution in DMF.6. Bromophenol blue: used as an indicator of coupling efficiency. Stock reagent is preparedby dissolving 33.5 mg of bromophenol blue in 5 mL of DMF. This should be diluted1:200 for working concentration.7. Acetylation mixture: DMF, acetic anhydride and triethylamine in a 50:5:1 (v/v/v) ratio.8. Side chain deprotection mixture: trifluoroacetic acid, ethanedithiol, and anisol in a 38:1:1 (v/v/v) ratio.9. Final wash solution: acetic acid 0.5% (v/v) in methanol/water (1:1, v/v).10. Other reagents: methanol (MeOH), purified water.2.3. ELISA Testing Procedure1. Phosphate buffered saline (PBS), 0.01 M, pH 7.2 (1.34 g of Na2HPO4·2H2O, 0.39 g ofNaH2PO4·2H2O, and 8.5 g of NaCl made up to 1 L with distilled water) is used as thebuffer base for most of the following buffer reagents.Table 1Suitable Amino Acid Side Chain Protecting Groupsfor Solid Phase Peptide Synthesis on PinsSide chain protecting group Amino acidt-Butyl ether S, T, Yt-Butyl ester D, Et-Butoxycarbonyl K, H, W2,2,5,7,8-Pentamethylchroman-6-sulfonyl RTrityl C Synthetic Peptides for Antimucin Antibodies 1352. Blocking buffer: 2% (w/v) BSA, 0.1% (v/v) Tween-20, and 0.1% sodium azide in 0.01 M PBS.3. Conjugate diluent: 1% (v/v) sheep serum, 0.1% (v/v) Tween-20, and 0.1% sodium casein-ate (USB, Bioscience, Cambridge, UK) in 0.01 M PBS.4. Citrate phosphate buffer: 17.8 g of Na2HPO4·2H2O and 16.8 g of citric acid monohydratemade up to 1 L with distilled water, pH 4.0.5. 2,2'-azino-bis[3-ethylbenz-thiazoline-6-sulfonic acid] (ABTS) substrate solution (Sigma):0.5 mg/mL in citrate phosphate buffer with hydrogen peroxide (35% w/w) added to givea final concentration of 0.01% (w/v).6. Disruption buffer: Sodium dihydrogen orthophosphate (0.1 M) pH 7.2, containing sodiumdodecyl sulfate (SDS) (0.1% w/v). β-Mercaptoethanol (5 mL) is added immediately prior to use.2.4. Purification of Antibodies Using Peptide Affinity Chromatography1. Affinity support: CNBr-activated Sepharose 4B (Pharmacia, Uppsala, Sweden).2. Equilibration buffer: 0.01 M PBS with azide (PBSA), pH 7.2 (1.34 g of Na2HPO4·2H2O,0.39 g of NaH2PO4·2H2O, and 8.5 g of NaCl made up to 1 L with distilled water) withsodium azide 0.02% (w/v) added as a preservative.3. Wash buffer: 0.5 M NaCl, pH 7.2, in distilled water.4. Elution buffer: 3 M NaSCN, pH 7.2, in distilled water.5. Desalting column: Sephadex G25 (Pharmacia).3. Methods3.1. Preparation of BSA-Peptide Conjugates (see Notes 1–3)1. Dissolve BSA (10 mg) in 3 mL conjugation buffer in a clean glass vial.2. Dissolve peptide (10 mg) in 1 mL conjugation buffer.3. To the BSA solution, add 1 mL of peptide solution and 10 µL of glutaraldehyde. Thenseal and agitate on a roller for 4 h at room temperature.4. The conjugate is finally dialysed against sodium chloride (1%) for 48 h at 4°C.3.2. Solid-Phase Peptide Synthesis on Pins (see Notes 4–11, and 17)The Multipin Peptide Synthesis Kit (Chiron Mimotopes) contains derivatized gears,stems, 8 × 12 format pin holders, and reaction trays. In addition, it contains all thesoftware needed for creating a synthesis schedule, running dispensing aids, and read-ing and plotting assay results.3.2.1. Creating a Synthesis ScheduleThe method described by Geysen et al. (8) for linear epitope scanning requires thatmany different peptides be synthesized simultaneously. To plan and execute a manualsynthesis schedule for creating hundreds of peptides simultaneously is extremely time-consuming and fraught with the possibility of errors. Fortunately, computer software isavailable to generate a synthesis schedule based on any given protein sequence with any ofthe manipulations described (Chiron Mimotopes). These schedules calculate the weightsand volumes of the various reagents required on each day of the synthesis and then instructthe operator where in the 96-well reaction tray each amino acid should go (see Notes 5–8).3.2.2. Peptide SynthesisThe peptide synthesis procedure consists of cycles of N-terminal deprotection,washing and coupling until the desired peptides have been assembled, followed by 136 Murray et al.side chain deprotection. The synthesis schedule provides details of the amounts ofamino acids and activators required. It is advisable to weigh out all these reagentsbefore beginning a synthesis since this is the most time-consuming step of the proce-dure. All steps are carried out at room temperature unless stated otherwise.1. The appropriate number of gears required for that synthesis on that day should be removedfrom storage and assembled on to the block according to the synthesis schedule. It is impor-tant that only gears requiring deprotection in the next cycle of synthesis be added to the block.2. Achieve deprotection of the amino terminus by immersing the pins in a bath containing20% piperidine for 20 min. The piperidine solution should cover the gears. The pins arethen washed as follows:a. DMF to cover gears for 2 min.b. MeOH (complete submersion) for 2 min.c. MeOH to cover gears for 2 min (three times).The pins are then allowed to air-dry in an acid-free fume hood for a minimum of 30 min.3. Prepare HOBt and DIC solutions by dissolving in the appropriate amount of DMF (seeNote 13). The addition of bromophenol blue to the HOBt to give a final concentration of0.05 mM as an indicator of coupling efficiency is optional. The volume of HOBt solutionspecified on the synthesis schedule must be added to each amino acid to dissolve it fullybefore adding the specified amount of DIC.4. Dispense amino acid solutions into a 96-well reaction tray according to the synthesisschedule. The recommended order of activating and dispensing amino acids is as follows:A D E F G I L M P S T V Y W Q N K C H R. Care should be taken to ensure that theamino acids are dispensed into the correct wells. Dispensing aids are now available thatconsist of a bank of LED lights set out in a microtiter plate format. Lights are lit beneaththe reaction tray to indicate which wells should contain which amino aid. The dispensingaid is driven by the synthesis schedule software.5. Place the block of Fmoc-deprotected pins into the reaction tray in the correct orientation.Place the tray into a polystyrene box to reduce evaporative losses and avoid contamina-tion and leave to incubate for at least 4 h.6. When the coupling reaction is complete, the blue colouration of bromophenol blue shouldhave disappeared. The pins are then washed as follows:a. MeOH to half the pin height for 5 min.b. air-dry for 2 min.c. DMF to half the pin height for 5 min.The next cycle of peptide synthesis can begin immediately with Fmoc deprotection.7. When the required peptides have been synthesized, deprotect and wash the free aminotermini as described in step 2. The amino terminus may then be acetylated to remove thecharge associated with a free amino terminus (if required) by incubating the pins in areaction tray containing acetylation mixture at 150 µL/well for 90 min in an enclosedenvironment. Then wash the pins in MeOH for 15 min and then air-dry.8. To achieve side chain deprotection, incubate the pins in a bath of side chain deprotectionmixture for 2.5 h. Next, immerse in a final wash solution for 1 h, rinse twice in MeOH for2 min each, and air-dry overnight. The pins are now ready for ELISA testing.3.3. ELISA Testing Procedure (see Notes 7 and 12)Antibody binding to peptides on pins is measured using an indirect ELISA proce-dure in which the solid phase on which the test antibody is captured is the peptide- Synthetic Peptides for Antimucin Antibodies 137coated gear, and the presence of the test antibody is reported using an enzyme-labeledsecondary antibody. The enzyme catalyzes the reaction of ABTS substrate to its col-ored product, which can be measured using a spectrophotometer. The degree of colorchange is proportional to the amount of test antibody bound to the peptide on the gear.Before antibody testing begins, the newly synthesized pins should be tested for non-specific binding to the enzyme-labeled secondary antibody of choice. This is achievedby carrying out steps 1 and 4–6. Antibodies may now be tested as follows, with allincubations and washing steps performed at room temperature unless otherwise stated.1. First precoat the pins in blocking buffer in order to minimize nonspecific binding to thegear. To achieve this, immerse the pins in a microtiter plate containing blocking buffer at200 µL/well and incubate for 1 h with agitation.2. Dilute the primary antibody to an appropriate concentration in blocking buffer and dispenseinto the wells of a microtiter plate at 200 µL/well. After removing from blocking buffer andflicking to remove excess buffer, incubate the pins in primary antibody at 4°C overnight.3. Remove the pins from the microtiter plate and wash four times in a bath of PBS contain-ing Tween-20 (0.1% v/v) for 10 min. Use fresh buffer for each wash.4. Dilute an appropriate horseradish peroxidase-labeled secondary antibody conjugate (e.g., horse-radish peroxidase-conjugated rabbit antimouse Ig is suitable for detecting murine primary anti-bodies) in conjugate diluent and dispense into the wells of a microtiter plate at 200 µL/well. Thenincubate the washed pins in the secondary antibody solution for 1 h with agitation.5. Wash the pins four times as in step 3. Prepare ABTS substrate solution immediatelybefore use and dispense into the wells of a microtiter plate at 200 µL/well. Immerse thepins in the substrate solution in the correct orientation and allow to incubate for 45 minwith agitation. The reaction can be stopped before the time is elapsed, if it appears thatthe reaction will give an optical density (OD) of 2 or greater, by removing the pins fromthe wells and then allowing the microtiter plate to shake for a further 15 min to allow fullcolor dispersion. The OD of each well is determined spectrophotometrically at a wave-length of 405 nm.6. Bound antibodies can be removed from the pins by sonication in disruption buffer at60°C for 2 h, followed by repeated rinses in distilled water at 60°C and methanol (twotimes). The efficiency of the cleaning procedure should be tested by repeating steps 4 and5. Absorbance levels above background indicate that antibody remains bound to the pinsand further cleaning is required. Once the pins are clean, they should be sonicated for 30min followed by rinsing as detailed just above (see Note 17).3.4. Purification of Antibodies Using Peptide Affinity Chromatography(see Notes 12–15 and Fig. 3)1. Prepare Sepharose-peptide affinity matrices and pack columns according to the manufacturer’sinstructions (see Notes 12 and 13).2. Equilibrate the columns with 10 column volumes of PBSA at a flow rate of 1 mL/min.3. Clarify hybridoma or bacterial culture supernatants by ultracentrifugation (40,000g, 1 h)and ultrafiltration (0.2 µm) and then store with 0.05% (w/v) sodium azide as a preservative.4. Apply clarified supernatant to the column at a rate of 1 mL/min followed by washing withPBSA, to remove unbound material, until the trace from the ultraviolet monitor has re-turned to baseline.5. Optional: Wash the column with 0.5 M NaCl (1 mL/min) to remove material that hasbound to the column nonspecifically. 138 Murray et al.6. To achieve desorption of specifically bound, pure antibody, apply three column volumesof 3 M NaSCN to the column at a rate of 1 mL/min and finally return the column to PBSA(see Notes 14 and 15).7. Antibody preparations desorbed using 3 M NaSCN must be desalted soon after elutionfrom the affinity matrix. To achieve this, connect a gel filtration column containing amedium such as Sephadex G25 in series with the affinity column (Fig. 3).4. Notes1. Conjugation of peptides to carrier proteins can also be performed in situ in the well of amicrotitier plate (11).2. Synthetic peptide-carrier protein conjugates and synthetic branched-chain polypeptidesprovide highly characterized and reproducible sources of mucin-like antigenic material.However, bear in mind that these reagents are analogs of the natural antigen and do notpossess the carbohydrate side chains that are a dominant characteristic of all mucins.Hence, results obtained in immunoassays, especially those involving the measurement ofkinetic data, must be treated with caution.3. The influence of carbohydrates on the recognition of peptide epitopes may be evaluated,at least to some extent, using synthetic glycopeptides rather than peptide alone. Glyco-peptides can be produced by both chemical (12) and enzymatic (13,14) methods. Thesereagents have been of value in assessing the contribution of O-linked N-acetyl-galactosamine (GalNAc) residues to mucin secondary structure and also in the studies toinvestigate the role of GalNAc residues in the binding of protein core antibodies (15).However, the glycosylation of mucin molecules is complex, and the production of higher-order synthetic mucin analogs with more than a single sugar at each glycosylation site istechnically demanding.4. The length of peptides synthesized seems to have no effect on the result obtained as dem-onstrated by two independent studies of anti-MUC1 protein core mAbs. One group usedheptamers spanning the tandem repeat domain and overlapping each other by six aminoacids (16), and the other used octamers overlapping each other by seven amino acids (17).Twelve antibodies were analyzed in total and the three that were analyzed in both studiesgave identical minimum binding units.5. In the omission analysis approach, a series of peptides are synthesized based on an epitopesequence. In each consecutive peptide, a single residue is omitted from the sequence.This allows the role of individual residues to be assessed. For example, an omission analy-sis series covering the immunodominant region of the MUC1 protein core may be synthe-sized. If an antibody were allowed to interact with this series of peptides, thosesequences that produced a loss in binding compared with the parent sequence can beidentified as part of a peptide in which an essential residue has been omitted. Antibodybinding is maintained in those peptides in which the epitope is complete.6. In the substitution analysis approach, each residue is replaced in turn with another aminoacid. This amino acid is normally alanine, but for cases in which an alanine already existsat that substitution position, the residue can be replaced with glycine. The informationprovided when an antibody is allowed to react with this set of peptides is similar to that ofomission analysis; however, in this case, the spatial arrangement of the respective epitoperesidues more closely resembles the native sequence.7. RNET analysis offers the most critical and informative method for probing a peptideepitope in the MUC1 protein core. In this approach, a set of peptides with sequencesbased on a short minimum binding sequence or epitope are synthesized on the heads of [...]... from the microtiter plate and wash four times in a bath of PBS contain- ing Tween-20 (0.1% v/v) for 10 min. Use fresh buffer for each wash. 4. Dilute an appropriate horseradish peroxidase-labeled secondary antibody conjugate (e.g., horse- radish peroxidase-conjugated rabbit antimouse Ig is suitable for detecting murine primary anti- bodies) in conjugate diluent and dispense into the wells of a microtiter... of therapeutic response in breast cancer using tumour mark- ers. Br. J. Cancer 64, 757–763. 4. Perkins, A. C., Symonds, I. M., Pimm, M. V., Price, M. R., Wastie, M. L., and Symonds, E. M. (1993) Immunoscintigraphy of ovarian carcinoma using a monoclonal antibody (In- 111-NCRC-48) defining polymorphic epithelial mucin. Nucl. Med. Commun. 14, 57 8-5 86. 5. Kunkler, R. B, Bishop, M. C., Green, D. J., Pimm,... by UDP-GalNAc:polypeptide N-acetylgalctosaminyltransferase from milk or mammary car- cinoma cells. Eur. J. Biochem. 229, 140–147. 15. Spencer, D. I. R., Price, M. R., Tendler, S. J. B., De Matteis, C. I., Stadie, T. and Hanisch, F G. (1996) Effect of glycosylation of a synthetic MUC1 mucin-core-related peptide on recognition by anti-mucin antibodies. Cancer Lett. 100, 11–15. 16. Price, M. R., Hudecz, F., O’Sullivan,... using an enzyme-labeled secondary antibody. The enzyme catalyzes the reaction of ABTS substrate to its col- ored product, which can be measured using a spectrophotometer. The degree of color change is proportional to the amount of test antibody bound to the peptide on the gear. Before antibody testing begins, the newly synthesized pins should be tested for non- specific binding to the enzyme-labeled secondary... A. (1994) N-Acetylgalactosamine glycosylation of MUC1 tandem repeat peptides by pancreatic tumour cell extracts. Cancer Res. 54, 3738–3744. Synthetic Peptides for Antimucin Antibodies 141 14. Stadie, T. R. E., Chai, W., Lawson, A. M., Byfield, P. G. H., and Hanisch, F-G. (1995) Studies on the order and site specificity of GalNAc transfer to MUC1 tandem repeats by UDP-GalNAc:polypeptide N-acetylgalctosaminyltransferase... reagents before beginning a synthesis since this is the most time-consuming step of the proce- dure. All steps are carried out at room temperature unless stated otherwise. 1. The appropriate number of gears required for that synthesis on that day should be removed from storage and assembled on to the block according to the synthesis schedule. It is impor- tant that only gears requiring deprotection in the next... binding to peptide epitopes con- jugated to synthetic branched chain polypeptide carriers. J. Immuol. Meth. 147, 201–210. 8. Geysen, H. M., Rodda, S. J., Mason, T. J., Tirbbick, G., and Schoofs, P. J. (1987) Strate- gies for epitope analysis using peptide synthesis. J. Immunol. Meth. 102, 259–274. 9. Price, M. R., Sekowski, M., and Tendler, S. J. B. (1991) Purification of anti-epithelial mucin monoclonal... chromatography. J. Immunol. Methods 139, 83–90. 11. Price, M. R., Sekowski, M., Hooi, D. S. W., Durrant, L. G., Hudecz, F., and Tendler, S. J. B. (1993) Measurement of antibody binding to antigenic peptides conjugated in situ to albumin coated microtitre plates. J. Immunol. Methods 159, 277–281. 12. Murray, A., Sekowski, M., Spencer, D. I. R., Denton, G. and Price, M. R. (1997) Purifica- tion of monoclonal... Fmoc-deprotected pins into the reaction tray in the correct orientation. Place the tray into a polystyrene box to reduce evaporative losses and avoid contamina- tion and leave to incubate for at least 4 h. 6. When the coupling reaction is complete, the blue colouration of bromophenol blue should have disappeared. The pins are then washed as follows: a. MeOH to half the pin height for 5 min. b. air-dry... air-dry. 8. To achieve side chain deprotection, incubate the pins in a bath of side chain deprotection mixture for 2.5 h. Next, immerse in a final wash solution for 1 h, rinse twice in MeOH for 2 min each, and air-dry overnight. The pins are now ready for ELISA testing. 3.3. ELISA Testing Procedure ( see Notes 7 and 12) Antibody binding to peptides on pins is measured using an indirect ELISA proce- dure . chain protecting group Amino acidt-Butyl ether S, T, Yt-Butyl ester D, Et-Butoxycarbonyl K, H, W2,2,5,7,8-Pentamethylchroman-6-sulfonyl RTrityl C Synthetic. monohydratemade up to 1 L with distilled water, pH 4.0.5. 2,2'-azino-bis[3-ethylbenz-thiazoline-6-sulfonic acid] (ABTS) substrate solution (Sigma):0.5 mg/mL