molecular diagnosis of infectious diseases, 2nd

M E T H O D S I N M O L E C U L A R M E D I C I N E TM Molecular Diagnosis of Infectious Diseases Second Edition Edited by Jochen Decker Udo Reischl Proteomic Approaches to Antigen Discovery Proteomic Approaches to Antigen Discovery Karen M Dobos, John S Spencer, Ian M Orme, and John T Belisle Abstract Proteomics has been widely applied to develop two-dimensional polyacrylamide gel electrophoresis maps and databases, evaluate gene expression profiles under different environmental conditions, assess global changes associated with specific mutations, and define drug targets of bacterial pathogens When coupled to immunological assays, proteomics may also be used to identify B-cell and T-cell antigens within complex protein mixtures This chapter describes the proteomic approaches developed by our laboratories to accelerate the antigen discovery program for Mycobacterium tuberculosis As presented or with minor modifications, these techniques may be universally applied to other bacterial pathogens or used to identify bacterial proteins possessing other immunological properties Key Words: Proteomics; antigen; B-cell; T-cell; Mycobacterium; bacteria; pathogens Introduction The ability to identify antigens produced by bacterial pathogens that are effective diagnostic or vaccine candidates of disease depends on multiple variables Two of the most important variables are the source of clinical specimens (immune T-cells or sera) used to identify potential antigens and the source or nature of the crude materials containing the putative antigens Factors that influence the usefulness of clinical specimens include whether samples were obtained from diseased individuals or experimentally infected animals and the state of disease at the time of specimen collection Likewise, the choice between native bacterial products and recombinant products as the starting material for antigen discovery efforts may significantly influence whether a useful antigen will be identified An equally important factor is the number of potential antigens that can be screened in a single experiment The use of recombinant molecular biology methods and the screening of large recombinant libraries is one approach toward maximizing the number of potential antigen targets (1–4) Although recombinant expression systems have been widely used for antigen identification, there From: Methods in Molecular Medicine, vol 94: Molecular Diagnosis of Infectious Diseases, 2/e Edited by: J Decker and U Reischl © Humana Press Inc., Totowa, NJ 01/Dobos/1-18/F 09/26/2003, 1:59 PM Dobos et al are several potential drawbacks Specifically, variability between the folding of a recombinant and native protein can complicate B-cell antigen discovery efforts (5), and the contamination of recombinant proteins with other bacterial products such as endotoxin is a major obstacle for cellular assays used to identify T-cell antigens (6) The ability to sequence whole genomes rapidly and the availability of several fully annotated bacterial genomes have profoundly altered the basic experimental approach to the study of bacterial physiology and pathogenesis (7,8) Previous to the sequencing of whole bacterial genomes, investigators would typically focus on a relatively small number of genes or gene products and develop specific assays to assess the activities or relevance of these gene products In contrast, the availability of whole genome sequences has now allowed for the development of methodologies such as DNA microarrays and proteomics to identify all the genes that are potentially involved in a specific cellular process (9–11) Unlike DNA microarrays, the technologies commonly used for proteomics studies [two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS) of peptides] have been around for decades (12,13) The power of these two technologies was brought together by the need to assess rapidly all the proteins produced in a particular bacterial species, as well as the development of innovative software that allows for the interrogation of MS data against genome sequences to identify proteins of interest (14) These technologies and the philosophy that we no longer need to focus on select sets of proteins, but should be evaluating the complete proteome in a single experiment can now be applied to antigen discovery efforts Moreover, proteomics technologies allow antigen discovery programs to focus on large sets of native proteins and eliminate a reliance on recombinant technologies to expand the pool of proteins to be screened In our laboratory, 2D-PAGE, Western blot analysis, and liquid chromatographymass spectrometry (LC-MS) were used to define 26 proteins of Mycobacterium tuberculosis that reacted with patient sera; three of these subsequently were determined to have significant potential as serodiagnostic reagents (15,16) This approach is a relatively facile method to screen for B-cell antigens The use of proteins resolved by 2D-PAGE and transferred to nitrocellulose was also applied to T-cell antigen identification (17) Although this work revealed several potential T-cell antigens, there are restrictions to its use In particular, the concentration of protein tested is unknown and the amount of protein obtained is most likely insufficient for multiple assays Thus, to increase the protein yield, we recently applied 2D liquid-phase electrophoresis (LPE) coupled with an in vitro interferon-γ (IFN-γ) assay and LC-tandem MS to identify 30 proteins from the culture filtrate and cytosol of M tuberculosis that possess a potent capacity to induce antigen-specific IFN-γ secretion from the splenocytes of M tuberculosis-infected mice (18) In this chapter, we detail the proteomics approach used in the identification of candidate B- and T-cell antigens from M tuberculosis However, these methods can be universally applied to the discovery of antigens from other bacterial pathogens as well as parasites 01/Dobos/1-18/F 09/26/2003, 1:59 PM Proteomic Approaches to Antigen Discovery Materials 2.1 Preparation of Subcellular Fractions Bacterial cell cultures (400 mL or greater) (see Note 1) Breaking buffer: phosphate-buffered saline (PBS; pH 7.4), mM EDTA, 0.7 µg/mL pepstatin, 0.5 µg/mL leupeptin, 0.2 mM phenylmethylsulfonyl fluoride (PMSF), 0.6 µg/mL DNase, µg/mL RNase (see Note 2) NaN3 Dialysis buffer: 10 mM ammonium bicarbonate, mM dithiothreitol (DTT), 0.02% NaN3 10 mM ammonium bicarbonate Vacuum pump Amicon ultrafiltration unit with a 10,000 Da MWCO membrane (Millipore, Bedford, MA; cat no PLGC07610) 0.2 µm Zap Cap S Plus bottle filtration units (Nalgene, Rochester, NY) Dialysis tubing (3500 Da MWCO) 10 French Press and French Press cell 11 Sterile 250-mL high-speed centrifuge tubes 12 Sterile 30-mL ultracentrifuge tubes 2.2 Identification of B-Cell Antigens via 2D Western Blot Analysis with Protein/Antigen Double Staining 2.2.1 Optimization of Serum Titers for Detection of Antigens by Western Blot 10 11 12 13 Human or experimental animal sera samples (see Notes and 4) 15% sodium dodecyl sulfate (SDS)-PAGE gels (7 × 10 cm) Protein molecular weight standards Laemmli sample buffer (5X): 0.36 g Tris-base, 5.0 mL glycerol, 1.0 g SDS, 5.0 mg bromophenol blue, 1.0 mL β-mercaptoethanol; QS to 10 mL with Milli-Q water, vortex, and store at 4°C or less (19) SDS-PAGE running buffer: 3.02 g Tris-base (pH 8.3), 14.42 g glycine, g SDS per L Nitrocellulose membrane, 0.22 µm Transfer buffer: 3.03 g Tris-base (pH 8.3), 14.4 g glycine (pH 8,3), 800 mL H2O, 200 mL CH3OH Dissolve reagents in water before adding CH3OH (20) Tris-buffered saline (TBS): 50 mM Tris-HCl (pH 7.4), 150 mM NaCl Wash buffer: TBS containing 0.5% vol/vol Tween 80 Blocking buffer: wash buffer containing 3% w/v bovine serum albumin (BSA) Anti-human IgG conjugated to horseradish peroxidase (HRP) BM Blue POD substrate, precipitating (Roche Molecular Biochemicals, Indianapolis, IN; cat no 1442066) Mini incubation trays for 2–4 mm nitrocellulose membrane strips (Bio-Rad, Hercules, CA; cat no 170-3902) 2.2.2 2D Western Blot Analysis for Identification of B-Cell Antigens Isoelectric focusing (IEF) rehydration buffer: M urea, 1% 3[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), 20 mM DTT, 0.5% ampholytes, 0.001% bromphenol blue (see Notes 5–7) Immobilized pH gradient (IPG) strips (21) (see Note 8) SDS-PAGE equilibration buffer: 150 mM Tris-HCl (pH 8.5), 0.2% SDS, 10% glycerol, 20 mM DTT, and 0.001% bromphenol blue 01/Dobos/1-18/F 09/26/2003, 1:59 PM Dobos et al 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 1% agarose dissolved in Milli-Q water Preparative SDS-PAGE gels (16 × 20 cm) Protein molecular weight standards SDS-PAGE running buffer (see Subheading 2.2.1., item 5) Coomassie stain: 1% coomassie brilliant blue R-250 in 40% methanol, 10% acetic acid (see Note 9) Coomassie destain 1: 40% methanol, 10% acetic acid Coomassie destain 2: 5% methanol Transfer buffer (see Subheading 2.2.1., item 7) Nitrocellulose, 0.22 µm Digoxigenin-3-O-methylcarbonyl-ε-aminocaproic acid-N-hydroxysuccinimide ester (DIG-NHS) (Roche Molecular Biochemicals; cat no 1333054); 0.5 mg/mL in N,Ndimethylformamide (DMF) 50 mM potassium phosphate buffer (pH 8.5) TBS: 50 mM Tris-HCl (pH 7.4), 150 mM NaCl Nonidet P-40, 10% solution H2O2, 30% solution Anti-digoxigenin-alkaline phosphatase, Fab fragments (Roche Molecular Biochemicals; cat no 1093274) INT/BCIP stock solution (Roche Molecular Biochemicals; cat no 1681460) INT/BCIP buffer: 100 mM Tris-HCl (pH 9.5), 50 mM MgCl2, 10 mM NaCl Anti-human IgG conjugated to HRP IPGphor IEF unit (Amersham Biosciences, Piscataway, NJ) or similar system SDS-PAGE electrophoresis unit Gel documentation system PDQuest 2D-gel analysis software (Bio-Rad) or similar software 2.2.3 Molecular Identification of Serum Reactive Proteins 0.2 M ammonium bicarbonate Trifluoroacetic acid (TFA; 10% solution) Destain solution: 60% acetonitrile, in 0.2 M ammonium bicarbonate Extraction solution: 60% acetonitrile, 0.1% TFA Modified trypsin, sequencing grade (Roche Molecular Biochemicals; cat no 1418025) Washed microcentrifuge tubes (0.65 mL) (see Note 10) Electrospray tandem mass spectrometer (such as LCQ classic, Thermo-Finnigan) coupled to a capillary high-performance liquid chromatography (HPLC) device Capillary C18-reverse phase (RP)-HPLC column Sequest software (14) for interrogating MS and MS/MS data against genomic or protein databases, or similar software 2.3 Identification of T-Cell Antigens 2.3.1 2D-LPE of Subcellular Fractions IEF protein solubilization buffer: M urea, mM DTT, 5% glycerol, 2% Nonidet P-40, and 2% ampholytes (pH 3.0–10.0 and pH 4.0–6.5 in a ratio of 1:4; see Note 7) Preparative SDS-PAGE gels (16 × 20 cm) SDS-PAGE running buffer (see Subheading 2.2.1., item 5) Laemmli sample buffer (see Subheading 2.2.1., item 4) 10 mM ammonium bicarbonate 01/Dobos/1-18/F 09/26/2003, 1:59 PM Proteomic Approaches to Antigen Discovery Rotofor preparative IEF unit (Bio-Rad) Whole Gel Eluter (Bio-Rad) 2.3.2 Assay of IFN-γ Induction for Identification of T-Cell Antigens Spleens from infected and naive mice (see Note 11) Complete RPMI medium (RPMI-1640 medium with L-glutamine, supplemented with 10% bovine fetal calf serum and 50 µM β-mercaptoethanol) (see Note 12) Hanks’ balanced salt solution (HBSS) Gey’s hypotonic red blood cell lysis solution: 155 mM NH4Cl, 10 mM KHCO3; use pyrogen-free water and filter-sterilize IFN-γ enzyme-linked immunosorbent assay (ELISA) assay kit (Genzyme Diagnostics, Cambridge, MA) (see Note 13) Concanavalin A (Con A) (see Note 14) 70-µm nylon screen (Becton/Dickinson, Franklin Lakes, NJ; cat no 35-2350) 96-well sterile tissue culture plates with lids 96-well microtiter ELISA plates, Dynex Immunlon (Dynex, Chantilly, VA) 10 Conical polypropylene centrifuge tubes (15 and 50 mL) 11 Sterile tissue culture grade Petri dishes (60 mm) 12 Syringes (3 mL) with 1-inch 22-gage needles 13 Hemocytometer 14 Tissue culture incubator (5% CO2, 37°C) 15 ELISA plate reader Methods 3.1 Preparation of Bacterial Subcellular Fractions (see Note 15) Grow bacterial cells to mid log phase The medium used should be devoid of exogenous proteins, such as BSA, that may interfere with 2D-PAGE analyses (see Note 1) Harvest cells by centrifugation at 3500g Decant the culture supernatant and save Wash the cell pellet with PBS (pH 7.4) and freeze until preparation of subcellular fractions (see Note 16) Add NaN3 to the culture supernatant at a final concentration of 0.04% (w/v) Filter the supernatant using a 0.2-µm filtration unit Concentrate the culture filtrate to 0.5% of its original volume using an Amicon ultrafiltration unit fitted with a 10-kDa MWCO membrane Dialyze the concentrated culture filtrate proteins (CFP) against dialysis buffer with at least two changes of this buffer, followed by a final dialysis step against 10 mM ammonium bicarbonate Filter-sterilize the dialyzed CFP using a 0.2-µm syringe filter or filtration unit Determine the protein concentration Store the final culture filtrate preparation at –80°C (see Note 17) 10 Place the frozen cell pellet at 4°C and thaw 11 Suspend the cells in ice-cold breaking buffer to a final concentration of g of cells (wet weight) per mL of buffer 12 Place the cell suspension in a French press cell and lyse via mechanical shearing by applying 20,000 psi of pressure with a French press Collect the lysate from the French press cell and place on ice (see Note 18) 13 To the lysate add an equal volume of breaking buffer and mix 01/Dobos/1-18/F 09/26/2003, 1:59 PM Dobos et al 14 Remove unbroken cells by centrifugation of the lysate at 3500g, 4°C in the tabletop centrifuge for 15 15 Collect the supernatant; this is the whole cell lysate 16 Further separation of the whole cell lysate into cell wall, cell membrane, and cytosol is achieved by differential centrifugation (22) First, centrifuge the lysate at 27,000g, 4°C for 30 Collect the supernatant and again centrifuge under the same conditions Collect the supernatant and store at 4°C 17 Suspend the 27,000g pellets in breaking buffer, combine them, and centrifuge at 27,000g, 4°C for 30 Discard the supernatant, and wash the pellet twice in breaking buffer The final 27,000g pellet represents the purified cell wall Suspend this pellet in 10 mM ammonium bicarbonate and extensively dialyze against 10 mM ammonium bicarbonate 18 Store the dialyzed cell wall suspension at –80°C, after estimating the protein concentration (see Note 17) 19 Add the supernatant collected in step 16 to ultracentrifuge tubes and centrifuge at 100,000g, 4°C for h 20 Collect the supernatant and centrifuge again under the same conditions 21 The final supernatant solution represents the cytosol fraction Dialyze the cytosol extensively against 10 mM ammonium bicarbonate, determine the protein concentration, and store at –80°C (see Note 17) 22 Suspend the 100,000g pellets obtained in steps 19 and 20 in breaking buffer, combine, and again centrifuge at 100,000g, 4°C for h This should be repeated twice 23 The final 100,000g pellet represents the total membrane Suspend the membranes in 10 mM ammonium bicarbonate, dialyze extensively against 10 mM ammonium bicarbonate, determine the protein concentration, and store at –80°C (see Note 17) 3.2 Identification of B-Cell Antigens via 2D Western Blot Analysis with Protein/Antigen Double Staining 3.2.1 Optimization of Serum Titers for Detection of Antigens by Western Blot Obtain and thaw a 100-µg aliquot (based on protein concentration) of the subcellular fraction to be analyzed One 100-µg aliquot will provide enough protein to optimize one patient’s serum or one sera pool and one matched control Dry the samples using a lyophilizer or speed vac Suspend the subcellular fraction in 80 µL PBS (pH 7.4) Add 20 µL of 5X Laemmli sample buffer to the sample and heat at 100°C for Apply 100 µL of sample to one preparative 15% SDS-polyacrylamide gel Resolve the proteins by 1D SDS-PAGE (19) After the electrophoresis is completed, assemble the gel into a Western blot apparatus and electrotransfer the proteins to a nitrocellulose membrane (20) Remove the nitrocellulose membrane and cut 2-mm vertical strips Place individual nitrocellulose strips in the wells of a mini incubation tray Add 500 µL of blocking buffer to each well and rock for h at room temperature (RT) or overnight at 4°C 10 Thaw the serum samples and generate dilutions (200 µL for each dilution) of the sera from 1:10 to 1:10,000 (see Note 19) 11 Incubate each strip with a single dilution of serum for h at RT with gentle agitation 12 Wash the strips repeatedly with 500 µL of wash buffer (minimally, five times) 13 Incubate the strips with 200 µL of the appropriate dilution of the anti-human IgG HRP for h at RT with gentle agitation 01/Dobos/1-18/F 09/26/2003, 1:59 PM Proteomic Approaches to Antigen Discovery 14 Wash the strips repeatedly with 500 µL of TBS (minimally, five times) 15 Develop the strips by addition of BM Blue POD substrate (200 µL, final volume per strip) 16 Stop the reaction by decanting the substrate and rinsing the strips with Milli-Q water 17 Determine optimum titer based on band intensity and background staining (see Note 20) 3.2.2 2D Western Blot Analysis for Identification of B-Cell Antigens Obtain and thaw 400-µg aliquots (see Note 17) of the subcellular fractions to be analyzed For each subcellular fraction or analysis, at least two aliquots of the selected subcellular fraction will be required, one for the 2D Western blot and one for a Coomassiestained 2D gel More aliquots will be required if replicate Western blots are to be performed or if comparisons between serum samples/pools are to be performed, such as a comparison between infected and healthy control serum Dry each aliquot of the subcellular fraction by lyophilization (see Note 21) Add 200 µL of rehydration buffer to each dried subcellular fraction and allow to stand at RT for h or at 4°C overnight Gentle vortexing can be applied if required Centrifuge the samples at 10,000g for 30 min, and remove the supernatant without disturbing the pellet (see Note 22) Transfer samples to IPG strips and allow the strips to rehydrate per manufacturer’s recommendations Resolve proteins by IEF (12) Remove strips from the IEF apparatus, and place in 16 × 150-mm glass test tubes with the acidic end of the strip near the mouth of the tube Apply 10 mL of SDS-PAGE equilibration buffer, and incubate at RT for 15 Warm 1% agarose solution while IPG strips are equilibrating Assemble 16 × 20-cm preparative SDS-PAGE gels into an electrophoresis apparatus Add running buffer to cover the lower half of the gels and inner core of the electrophoresis apparatus 10 Remove IPG strips, clip ends of IPG strips (where no gel is present), and guide each strip into the well of the preparative SDS-PAGE gels using a flat spatula or small pipet tip The acidic end of the IPG strip should be next to the reference well for the molecular weight standards 11 Overlay each strip with 1% agarose When adding agarose, move from one end of the strip to the other This helps prevent the trapping of air bubbles between the strip and interface of the SDS-PAGE gel Use enough agarose to cover the strip fully 12 Add molecular weight standards to the reference well of each gel 13 Resolve proteins in the second dimension by electrophoresis (19) 14 Remove the preparative SDS-PAGE gels Stain one gel with Coomassie (see Note 9) 15 After staining, use a gel documentation system to scan a tif image of the gel Ensure that the gel is scanned under parameters compatible with the 2D gel analysis software that will be used 16 Place the gel in a storage tray, cover with Milli-Q water, seal, and store at 4°C 17 Assemble the second gel into a Western blot apparatus and electrotransfer the proteins to a nitrocellulose membrane (20) 18 Wash nitrocellulose membrane five times with 20 mL of 50 mM potassium phosphate (pH 8.5) 19 Prepare total protein labeling solution by adding 10 µL of DIG-NHS and 20 µL of 10% Nonidet P-40 to 20 mL of 50 mM potassium phosphate buffer (pH 8.5) 01/Dobos/1-18/F 09/26/2003, 1:59 PM 10 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 Dobos et al Incubate membrane in labeling solution for h at RT with gentle agitation Wash the membrane five times in 20 mL of TBS Incubate the labeled membrane in blocking buffer for h at RT with gentle agitation Wash the membrane briefly with TBS Add serum at the titer optimized in Subheading 3.2.1 and incubate at RT for h with gentle agitation Wash the membrane five times with 20 mL TBS Add 20 µL of anti-digoxigenin-alkaline phosphatase to 20 mL of TBS and incubate the membrane in this solution for h with gentle agitation Wash the membrane five times with 20 mL of TBS Add anti-human IgG HRP diluted per manufacturer’s instructions into TBS, and incubate the membrane in this solution for h at RT with gentle agitation Wash the membrane five times with 20 mL of TBS Rinse the membrane briefly in Milli-Q water To visualize the serum-specific antigens, incubate the membrane in 10 mL of BM Blue POD substrate without agitation Watch the membranes for blue/purple color development Aspirate the substrate as soon as color develops, and rinse briefly with Milli-Q water Using a gel documentation system, capture a tif image of the Western blot showing the serum reactive proteins To visualize the total protein profile on the Western blot, generate the alkaline phosphatase substrate by adding 75 µL of INT/BCIP stock solution to 10 mL of INT/BCIP buffer Add to the membrane, incubate without agitation, and watch for color development Aspirate the substrate solution when reddish brown spots are well defined, and rinse the membrane briefly with Milli-Q water Using a gel documentation system, capture a tif image of the Western blot showing the total protein profile Transfer the images of the Coomassie-stained gel, the serum reactive proteins, and the total protein profile of the 2D Western blot to a 2D analysis program Using this program, match the spots of the three images to allow for identification of the protein spots within the Coomassie-stained 2D gel that correspond to the serum reactive proteins 3.2.3 Molecular Identification of Serum Reactive Proteins From the Coomassie-stained 2D-gel, excise the protein spots corresponding to those reactive to serum on the 2D Western blot Cut each gel slice into small pieces (1 × mm), and place the gel pieces from each spot in separate washed microcentrifuge tubes Destain by covering the gel pieces with destain solution, and incubate at 37°C for 30 Discard the acetonitrile solution and repeat step until the gel slices are completely destained Dry the gel pieces under vacuum Dissolve 25 µg of modified trypsin in 300 µL of 0.2 M ammonium bicarbonate Add 3–5 µL of the trypsin solution to the gel slices Incubate at room temperature until the trypsin solution is completely absorbed by the gel, approx 15 Add 0.2 M ammonium bicarbonate in 10–15 µL increments to rehydrate the gel pieces completely Allow about 10 for each aliquot of ammonium bicarbonate to be absorbed by the gel Also avoid adding an excess of ammonium bicarbonate solution 10 Incubate the gel slices for 4–12 h at 37°C 01/Dobos/1-18/F 10 09/26/2003, 1:59 PM Proteomic Approaches to Antigen Discovery 11 11 12 13 14 15 Terminate the reaction by adding 0.1 vol of 10% TFA Collect the supernatant, and place it in a new washed microcentrifuge tube Add 100-µL of the extract solution to the gel slices and vortex Incubate the extract solution and gel slices at 37°C for 40 Centrifuge the extract, collect the supernatant, and add it to the supernatant collected in step 12 16 Repeat steps 13–15 17 Dry the extract under vacuum 18 Store the dried peptide extracts at –20°C until analysis by LC-MS/MS (14) 3.3 Identification of T-Cell Antigens (see Note 23) 3.3.1 2D-LPE of Subcellular Fractions Obtain and thaw 250-mg aliquots of the subcellular fraction(s) to be analyzed Dry the subcellular fraction by lyophilization (see Note 21) Solubilize the proteins by adding 60 mL IEF rehydration buffer, and incubate at RT for h or at 4°C overnight Centrifuge the suspended material at 27,000g to remove particulates (see Note 22) Collect the supernatant and apply this material to the Rotofor (Bio-Rad) apparatus per the manufacturer’s instructions Preparative IEF of the sample should be performed at a constant power of 12 W until the voltage stops increasing and stabilizes (see Note 24) Harvest the samples from the Rotofor per manufacturer’s instructions Evaluate 8–10 µL of each preparative IEF fraction by SDS-PAGE and Coomassie staining Pool those fractions that have a high degree of overlap in their protein profile as observed by SDS-PAGE (see Note 25) Dialyze the IEF fractions extensively against ammonium bicarbonate After dialysis determine the protein concentration 10 Split the IEF fractions into 5-mg aliquots and dry by lyophilization or speed vac 11 To separate each IEF fraction in the second dimension, solubilize mg of each fraction in 1.6 mL of PBS and add 0.4 mL of 5X Laemmli sample buffer 12 Apply each fraction to the preparative well of a 16 × 20-cm SDS-PAGE gel (see Note 26) 13 Resolve proteins in the second dimension by electrophoresis 14 Remove polyacrylamide gels from glass plates and soak in 100 mL of 10 mM ammonium bicarbonate for 30 with one change of the 10 mM ammonium bicarbonate 15 While the gel is equilibrating in 10 mM ammonium bicarbonate, assemble the Whole Gel Eluter (Bio-Rad) per manufacturer’s instructions and fill the Whole Gel Eluter wells with 10 mM ammonium bicarbonate (see Note 27) 16 Cut SDS-PAGE gel to the dimension of the Whole Gel Eluter 17 Lay the SDS-PAGE gel on top of Whole Gel Eluter wells Orientate the gel so that protein bands run parallel to the wells 18 Complete the setup of the Whole Gel Eluter per manufacturer’s instructions, and elute the proteins at 250 mA for 90 19 At the end of the elution, reverse the current on the Whole Gel Eluter for 20 s 20 Harvest the samples from the Whole Gel Eluter per manufacturer’s instructions, This will yield 30 fractions of approximately 2.5 mL each 21 Filter sterilize each fraction with a 0.2-µm PTFE syringe filter (Use aseptic techniques for subsequent manipulation of the 2D-LPE fractions.) 22 Determine the protein concentration of each fraction 23 Split each fraction into 10-µg aliquots, lyophilize, and store at –80°C 01/Dobos/1-18/F 11 09/26/2003, 1:59 PM 442 Winger Fig 11 Telemedicine Information Exchange One platform particularly favored by universities (including the University of Newcastle) is the offering by http://www.blackboard.com These electronic classrooms could be a useful advance for central diagnostic laboratories that have satellite stations 2.11 Telemedicine Connections Telemedicine can be roughly defined as an electronic-based communication between two remote locations for clinical diagnostic purposes In this sense, telemedicine can be an important tool in the clinical diagnostic sphere and will certainly increase in use as centralized laboratories need to communicate with far-distant outposts Telemedicine Information Exchange, a service of America’s National Library of Medicine, provides exhaustive information on all events relating to telemedicine, worldwide (http://tie.telemed.org) (Fig 11) 2.12 Time Wasters Some Internet areas are not worthwhile or are counterproductive for the clinical or research diagnostics specialist (newsgroups, chatrooms, email spam) or are of little usefulness (electronic conferences and virtual reality rooms) What a disappointment the UseNet newsgroups have turned out to be! Five years ago, these internet bulletin boards held the promise of fast and immediate access to the 24/Winger/429-444/F 442 09/26/2003, 4:47 PM Molecular Diagnostics Resources on the Internet 443 expertise of a world community dedicated to a particular topic Today most are moribund repositories of tired old rehashed arguments or endless lists of favorites, or the site of intensely vituperative flame wars among angst-ridden or blithely guilt-free individuals Not a place for dedicated diagnostic professionals The harvesting of email information from these sites by spammers (mass email advertisers) was one of the death knells of this potentially useful medium, and this practice is more than sufficient inducement to keep careful professionals away, particularly if, as itemized below, you are in a politically turbulent area of work If you are a fast typist, chatrooms have an immediacy that can be seductive, but who really wants to communicate in real time through their fingers? These areas are ideal for lonely hearts or teenage romance, but it’s difficult to see how they can be useful apart from the very restricted practice of electronic classrooms (as above) Pick up the phone and convey much more information directly from mouth to ear! Or, if convenience between correspondents is the goal (and it is), an email can be as immediate as a chatroom but with less capacity than the phone to intrude on other more pressing involvements Electronic conferences and other virtual reality spaces are ideas whose time has not yet come in the molecular diagnostic resource sphere Although pure chemists seem to enjoy the utility of electronic conferencing, by which displays of virtual molecules can be conveniently disseminated, similar activities not seem to be organized with the clinical diagnosis field in mind Perhaps this is because Clinical Chemistry and Clinical Diagnosis conferences tend to be financed by the major clinical chemistry/ immunoassay platforms, so that the actual, physical interaction of conference participants is, in effect, subsidized Certainly electronic conferencing will need to improve by several orders of magnitude before the amount of information exchange can begin to approximate that available on a real conference floor 2.13 Staying Visible or Nonvisible; Ensuring and/or Safeguarding Your Own Presence, or Nonpresence, on the Net If you are in academia, your institution will have ensured that your presence in the community is noted, your research interests itemized, and your publication list presented for all to see Alternatively, you will have been pushing for your own specific website demarcating the research interests of your laboratory Similarly, if you happen to be working in the commercial sphere, your company will undoubtedly have a web presence touting for custom, advertising your particular diagnostic wares, skill base, and technology/machinery In these circumstances, you will probably wish to have a high profile, and you can help to ensure high visibility by informing the search engines of your web pages, by spreading the address of your website around so that interested browsers are constantly reminded of your site (Email signature files are a simple and highly effective way to identify yourself and your site.) Alternatively, you may be part of a commercial team or a clinical research institution that wishes to remain beneath the threshold of visibility If you harvest serum from immunized animals, for example, or are involved in the daily generation of mono- 24/Winger/429-444/F 443 09/26/2003, 4:47 PM 444 Winger clonal antibodies, you will know of the potential ramifications of these practices relative to the activities of today’s animal-rights activists In such circumstances, you may wish to be protected from high visibility in the world-wide domain of the net Your computer systems’ administrator will undoubtedly set protocols of security as a matter of course, if you are in such a situation Common sense adherence to the demands of your company or institution (whether they are as painless as a signature file that disclaims any institutional authority for your message, or as fundamental as a multilayered firewall-protected system through which your browser must traverse before any downloaded files reach your computer) will help to ensure that these security demands are met Conclusions The Internet is a fundamental component of life for busy scientists and clinical practitioners in the diagnostic sphere The information resources available can prevent the loss of countless hours in fruitless attempts to “reinvent the wheel.” Alternatively, specific information can identify an important tool that could mean the difference between success and failure in the performance of a particular assay Wise and careful use of this global information conduit that connects diagnostic researchers world-wide must be a matter of daily bread-and-butter activity by any conscientious participant in this exciting field 24/Winger/429-444/F 444 09/26/2003, 4:47 PM Cloning scFv from Hybridoma Cells 447 25 Cloning Single-Chain Antibody Fragments (scFv) from Hybridoma Cells Lars Toleikis, Olaf Broders, and Stefan Dübel Abstract Despite the availability of antibody libraries for the selection of receptor molecules, the large number of established and well-characterized hybridoma lines still represent a useful source for recombinant antibody genes This protocol describes the PCR amplification, cloning, and a small-scale expression test for the generation of scFv fragments from hybridoma cell lines Particular emphasis was placed on frequently observed problems and pitfalls of this method Key Words: Antibody engineering; proteomics; monoclonal antibodies; phage display Introduction The variable region (Fv) portion of an antibody is comprised of the antibody VH and VL domains and is the smallest antibody fragment containing a complete antigen binding site To stabilize the fragment kinetically by generating a very high local concentration for the association of the recombinant VH and VL domains, they are usually linked in single-chain Fv (scFv) constructs with a short peptide that bridges the approx 3.5 nm between the carboxy-terminus of one domain and the amino-terminus of the other (1–3) The cloning of these variable domain genes has been well established as a common method for the “immortalization” of valuable mouse or rat hybridoma clones Furthermore, a large number of protein fusions to the antigen binding variable (Fv) portion of an antibody in E coli has been constructed to add a variety of heterologous functions The genetic information for the variable heavy and light chain domains (VH and VL) is generally amplified from hybridoma cells using polymerase chain reaction (PCR) with immunoglobulin-specific primers A variety of primer sets for the amplification of mouse variable domains have been developed (4–6) However, several different sequences for each domain may be found in the PCR products From: Methods in Molecular Medicine, vol 94: Molecular Diagnosis of Infectious Diseases, 2/e Edited by: J Decker and U Reischl © Humana Press Inc., Totowa, NJ 447 25/Toleikis/445-458/F 447 09/26/2003, 4:47 PM 448 Toleikis, Broders, and Dübel amplified from the cDNA of a single hybridoma “clone.” The fact that one cell line might express more than one heavy and light chain was also observed by other authors (7–10) Up to nine different VL and five different VH sequences with a homology of about 95% have been isolated from a single hybridoma culture of hybridoma cell line PA-1 (S Deyev, personal communication) Various reasons may be responsible for these heterogenous results Additional antibody variable domain genes may derive from antibody mRNA transcribed from the genes of the parental cells, e.g., the myeloma fusion partner used for generation of the hybridoma line These chains, found in older fusion lines, are the easiest to identify by comparison with the known sequences of the original fusion partners Allelic dysregulation may lead to the expression of all V regions available, and pseudogenes with an internal stop codon were found as the most abundant PCR product in some cases Even tandem-like assemblies of parts of two different V region sequences have been observed (J Görnemann, personal communication) It has to be kept in mind that hybridoma cells are very different from the well-regulated antibodyproducing cells in our body In addition to their doubled ploidy and partial cancer cell origin, they are completely released from the strict regulation of the immune system, allowing for an abundance of gene expression deviations Mutations may be introduced into the antibody genes during prolonged cell culture, resulting in a heterogenous population containing a set of highly homologous antibody genes Therefore, it is very important to prepare the cDNA from a freshly subcloned hybridoma clone tested for productivity Furthermore, mutations may be introduced in the PCR amplification step However, the use of polymerase with proofreading activity should suppress these mutations Further sequence variation is frequently introduced by the use of degenerated mixtures of oligonucleotide primers (6) This cannot be avoided completely since it is not possible to use primer sets matching 100% with all possible sequences, simply because of the observed variation in the framework region and the fact that not all somatic antibody sequences can be known Furthermore, owing to the necessity of amplifying unknown sequences, the hybridization conditions during PCR of antibody sequences from cDNA have to be adjusted to allow mismatches Under these conditions, even amplification of antibody DNA belonging to several different subgroups with the same primer was observed (10) Particular attention has to be paid to the position of VH, where a mutational toggling between Gln and Glu has been shown to influence the stability of the product dramatically (11,12) As a result, the common strategy should be to analyze quite a number of randomly picked VH and VL clones after the PCR, in a first screen for heterogenicity A quick method is to compare the restriction patterns after digestion with BstNI (13) This enzyme has been shown to cleave different sites in V regions frequently It is not completely conclusive, because it may give a hint that V-region genes of different genetic origin are present, but it will rarely detect point mutations It is more advisable to sequence the DNA of at least 10–20 clones of each heavy and light chain PCR reaction product If different sequences are found, their various combinations should be assembled in an expression vector and tested for antigen binding separately 25/Toleikis/445-458/F 448 09/26/2003, 4:47 PM Cloning scFv from Hybridoma Cells 449 A protein sequencing step (preferentially of tryptic peptides) may be employed to identify the correct sequence Peptide sequences containing complementarily-determining regions (CDRs) or their fragments may serve as a template for designing specific oligonucleotide primers for a PCR assembly cloning of the correct sequence in case no product or only incorrect products are amplified by the available sets of primers In case all directly cloned combinations of VH and VL fail to bind, the construction of a small phage library from each hybridoma by cloning the PCR products into a surface expression phagemid (e.g., pSEX; see refs 14 and 15) is recommended This hybridoma library may then be screened for binding activity by panning on antigen Because in this approach all the possible combinations of all sequences derived from the hybridoma cDNA are directly screened for antigen binding (16), wrong sequences, wrong VH/VL pairs, and ligation artifacts are excluded However, even if the correct sequences have been cloned, an scFv may fail to bind in an assay optimized for the original monoclonal for several reasons First, expression levels of individual scFvs derived from hybridomas can vary dramatically depending on the individual V-region sequences, mostly as a result of their folding efficiency, with the consequence of impractically low yields in some cases (17) The periplasmic preparation used for the specificity test should therefore always be analyzed for its content of scFvs, most conveniently by immunoblot using an anti-tag reagent Second, even though many scFvs are quite rigid molecules, others may be unstable in solution, their activity decaying within a few hours In contrast to scFvs derived from phage display libraries (which during the panning rounds in addition to specificity are coselected for efficient functional expression in E coli), hybridomaderived scFv fragments have not undergone this selection The use of freshly prepared periplasmic fractions can help to prove specificity of such a sequence prior to a genetic stabilization by subcloning the V regions into vectors allowing expression as Fab or IgG Finally, scFvs designed in the usual regime (with a 15–18 amino acid linker connecting the two regions) may fail to bind in comparison with their monoclonal parent owing to the loss of the avidity effect allowed by densely coated antigen With (in case of IgG) or 10 (in case of IgM) binding arms, the apparent affinity of multivalent molecules in enzyme-linked immunosorbent assay (ELISA) or on an Immunoblot may be increased by orders of magnitude compared with a single binding domain The best way to check this would be to use Fab fragments of the parental monoclonal antibody (MAb) for positive control However, these are not always easily prepared, but the vice versa approach can be tried by dimerizing/multimerizing the scFv A straightforward approach to achieve this is reduction of the linker length between the VH and VL, thus forcing the scFv to assemble in the diabody/triabody/tetrabody format Alternatively, one of various vectors encoding multimerization domains fused downstream to the scFv gene can be used (for review, see ref 18) Finally, a number of scFv with post-translational modifications in the CDR regions have been described Unpaired cysteines in a CDR may affect the activity, in particular upon long-term storage, as they are prone to oxidation (19) Glycosylation is frequently found in various CDRs, but it does not necessarily affect binding (20) In some cases, the glycosylation decreases the affinity and the antibody benefits from 25/Toleikis/445-458/F 449 09/26/2003, 4:47 PM 450 Toleikis, Broders, and Dübel a mutation removing the glycosylation site (21,22) However, when the sugars contribute to the binding, the scFvs may not be functionally produced in E coli Mammalian cell lines [e.g., Chinese hamster ovary (CHO)] are the expression host of choice in these cases The contribution of sugars to the binding may be analyzed by glycosidase digestion of the maternal hybridoma antibody A large variety of expression vectors for scFv fragments have been developed They all allow a secretion of the scFv fragment into the periplasmic space of E coli, where the biochemical milieu promotes correct folding and formation of the intrachain disulfide bonds This is achieved by employing an amino-terminal bacterial leader sequence (23), which is removed during secretion Most vectors introduce additional motifs suitable for identification and/or purification of the scFv fragments Most common is the His tag, allowing both IMAC purification and detection on Immunoblots, in ELISA, and so on An example is the vector pOPE101 (24) Some vectors encode an unpaired Cys residue in the tag region of the expressed polypeptide These vectors should only be employed if the cysteine residue is required afterward for chemical conjugation because unpaired Cys residues have a negative influence on the yield (24) A collection of complementing protocols on various recombinant antibody selection, expression, and analysis systems as well as alternative hybridoma cloning methods can be found in ref 25 Materials 2.1 Isolation of Antibody DNA RNeasy Mini Kit for total RNA minipreps (Qiagen, Hilden, Germany) QIAshredder Homogenizer Kit (Qiagen) Oligotex mRNA Mini Kit for isolation of mRNA from total RNA (Qiagen) SuperScript™ First-Strand Synthesis System for RT-PCR (Invitrogen, Karlsruhe, Germany) DNA polymerase with proofreading function, e.g., ProofStart DNA Polymerase (Qiagen) or Expand High Fidelity PCR System (Roche, Mannheim, Germany) dNTP Mix (MBI Fermentas, St Leon-Rot, Germany) Thermal cycler Agarose gel electrophoresis equipment QIAquick PCR Purification Kit and QIAquick Gel Extraction Kit (Qiagen) 2.2 Cloning into an Expression Vector Restriction endonucleases HindIII, MluI, NcoI, and NotI with appropriate buffers (New England Biolabs, Frankfurt, Germany) Alkaline phosphatase (CIP) for DNA dephosphorylation (Roche) pOPE101 expression vector (24) Agarose gel electrophoresis equipment QIAquick PCR Purification Kit and QIAquick Gel Extraction Kit (Qiagen) T4 DNA ligase and buffer (Invitrogen) Electroporation equipment n-Butanol (1- or 2-butanol will do) E coli XL1-Blue electroporation-competent cells (Stratagene, Amsterdam, The Netherlands) 10 Luria–Bertani (LB) medium containing 100 mM glucose and 100 µg/mL ampicillin 11 LB agar plates containing 100 mM glucose and 100 µg/mL ampicillin 25/Toleikis/445-458/F 450 09/26/2003, 4:48 PM Cloning scFv from Hybridoma Cells 451 12 QIAprep Spin Miniprep Kit (Qiagen) 13 Glycerol 2.3 Small-Scale Expression Test LB medium containing 100 mM glucose and 100 µg/mL ampicillin 100 mM solution of isopropyl-β-D -thiogalactopyranoside (IPTG) Store in aliquots at –20°C in the dark Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot equipment Anti-c-myc monoclonal antibody Myc1-9E10 (Invitrogen) and/or anti-penta-His monoclonal antibody (Qiagen) Horseradish peroxidase (HRP)-conjugated goat-anti-mouse IgG antibody (Invitrogen) Tetramethylbenzidine (TMB)-stabilized substrate for HRP (Progen, Heidelberg, Germany) Methods Methods that are not specifically described step by step can be done according to the manufacturers’ protocols (Kits) or according to Sambrook et al (26) 3.1 Isolation of Antibody DNA Collect up to × 106 hybridoma cells by centrifugation Isolate the total RNA from the hybridoma cells using a kit (see Note 1) Isolate the mRNA from the total RNA using a kit (see Note 2) Prepare the first-strand cDNA from the mRNA using a kit (see Note 3) A second-strand synthesis is not necessary First PCR for the amplification of antibody DNA: For each reaction combine the primer specific for the constant domain and a primer specific for the variable domain (Table 1) Perform each reaction in a total volume of 50 µL Each 50-µL reaction contains 25 pmol of each primer, dNTP mix (10 mM of each), and polymerase buffer as described by the supplier Use µL of the prepared cDNA for each 50-µL reaction Add µL (2.5 U) of ProofStart or High Fidelity DNA Polymerase per 50 µL reaction (see Note 4) Place the PCR tubes in a thermal cycler (see Note 5) Denature at 95°C for (see Note 6) Perform 30 cycles with the following cycling program: 45 s of denaturation at 94°C, 45 s of annealing at the appropriate primer hybridization temperature (see Note 7), and 90 s of extension at 72°C After 30 cycles, perform a final extension step for at 72°C Take out 1/10 volume of each PCR reaction for analytical gel electrophoresis on a 2% agarose gel Purify the PCR products using a kit Elute DNA in H2O (see Note 8) Second PCR for reamplification and introduction of appropriate restriction sites into the antibody DNA: 10 Each 50-µL reaction contains 25 pmol of each primer, dNTP mix (10 mM each), and polymerase buffer as described by the supplier Use up to 50 ng of purified PCR product from the first PCR for each 50-µL reaction Add µL (2.5 U) of ProofStart or High Fidelity DNA Polymerase per 50 µL reaction (see Note 4) Use the primer pairs (with the introduced restriction sites; see Table 2) that gave the desired PCR product in the first PCR 25/Toleikis/445-458/F 451 09/26/2003, 4:48 PM 452 Toleikis, Broders, and Dübel Table Oligonucleotides for the Amplification of Mouse Immunoglobulin Variable Region DNA Heavy chain Constant domain MHC.F 5'-GGCCAGTGGATAGTCAGATGGGGGTGTCGTTTTGGC-3' Variable domain MHV.B1 MHV.B2 MHV.B3 MHV.B4 MHV.B5 MHV.B6 MHV.B7 MHV.B8 MHV.B9 MHV.B10 MHV.B12 5'-GATGTGAAGCTTCAGGAGTC-3' 5'-CAGGTGCAGCTGAAGGAGTC-3' 5'-CAGGTGCAGCTGAAGCAGTC-3' 5'-CAGGTTACTCTGAAAGAGTC-3' 5'-GAGGTCCAGCTGCAACAATCT-3' 5'-GAGGTCCAGCTGCAGCAGTC-3' 5'-CAGGTCCAACTGCAGCAGCCT-3' 5'-GAGGTGAAGCTGGTGGAGTC-3' 5'-GAGGTGAAGCTGGTGGAATC-3' 5'-GATGTGAACTTGGAAGTGTC-3' 5'-GAGGTGCAGCTGGAGGAGTC-3' Light chain Kappa chain constant domain MKC.F 5'-GGATACAGTTGGTGCAGCATC-3' Kappa chain variable domain MKV.B1 MKV.B2 MKV.B3 MKV.B4 MKV.B5 MKV.B6 MKV.B7 MKV.B8 MKV.B9 MKV.B10 5'-GATGTTTTGATGACCCAAACT-3' 5'-GATATTGTGATGACGCAGGCT-3' 5'-GATATTGTGATAACCCAG-3' 5'-GACATTGTGCTGACCCAATCT-3' 5'-GACATTGTGATGACCCAGTCT-3' 5'-GATATTGTGCTAACTCAGTCT-3' 5'-GATATCCAGATGACACAGACT-3' 5'-GACATCCAGCTGACTCAGTCT-3' 5'-CAAATTGTTCTCACCCAGTCT-3' 5'-GACATTCTGATGACCCAGTCT-3' Lambda chain constant domain MLC.F 5'-GGTGAGTGTGGGAGTGGACTTGGGCTG-3' Lambda chain variable domain MLV.B 5'-CAGGCTGTTGTGACTCAGGAA-3' Data from ref 27 452 25/Toleikis/445-458/F 452 09/26/2003, 4:48 PM Cloning scFv from Hybridoma Cells 453 Table Oligonucleotides for the Reamplification of Mouse Immunoglobulin Variable Region DNA and Introduction of Appropriate Restriction Sitesa Heavy chain Constant domain MHC.F.Hind 5'-GGCCAGTGGATAAAGCTTTGGGGGTGTCGTTTTGGC-3' Variable domain MHV.B1.Nco MHV.B2.Nco MHV.B3.Nco MHV.B4.Nco MHV.B5.Nco MHV.B6.Nco MHV.B7.Nco MHV.B8.Nco MHV.B9.Nco MHV.B10.Nco MHV.B12.Nco 5'-GAATAGGCCATGGCGGATGTGAAGCTGCAGGAGTC-3' 5'-GAATAGGCCATGGCGCAGGTGCAGCTGAAGGAGTC-3' 5'-GAATAGGCCATGGCGCAGGTGCAGCTGAAGCAGTC-3' 5'-GAATAGGCCATGGCGCAGGTTACTCTGAAAGAGTC-3' 5'-GAATAGGCCATGGCGGAGGTCCAGCTGCAACAATCT-3' 5'-GAATAGGCCATGGCGGAGGTCCAGCTGCAGCAGTC-3' 5'-GAATAGACCATGGCGCAGGTCCAACTGCAGCAGCCT-3' 5'-GAATAGGCCATGGCGGAGGTGAAGCTGGTGGAGTC-3' 5'-GAATAGGCCATGGCGGAGGTGAAGCTGGTGGAATC-3' 5'-GAATAGGCCATGGCGGATGTGAACTTGGAAGTGTC-3' 5'-GAATAGGCCATGGCGGAGGTGCAGCTGGAGGAGTC-3' Light chain Kappa chain constant domain MKC.F.Not 5'-TGACAAGCTTGCGGCCGCGGATACAGTTGGTGCAGCATC-3' Kappa chain variable domain MKV.B1.Mlu MKV.B2.Mlu MKV.B3.Mlu MKV.B4.Mlu MKV.B5.Mlu MKV.B6.Mlu MKV.B7.Mlu MKV.B8 Mlu MKV.B9.Mlu MKV.B10.Mlu 5'-TACAGGATCCACGCGTAGATGTTTTGATGACCCAAACT-3' 5'-TACAGGATCCACGCGTAGATATTGTGATGACGCAGGCT-3' 5'-TACAGGATCCACGCGTAGATATTGTGATAACCCAG-3' 5'-TACAGGATCCACGCGTAGACATTGTGCTGACCCAATCT-3' 5'-TACAGGATCCACGCGTAGACATTGTGATGACCCAGTCT-3' 5'-TACAGGATCCACGCGTAGATATTGTGCTAACTCAGTCT-3' 5'-TACAGGATCCACGCGTAGATATCCAGATGACACAGACT-3' 5'-TACAGGATCCACGCGTAGACATCCAGCTGACTCAGTCT-3' 5'-TACAGGATCCACGCGTACAAATTGTTCTCACCCAGTCT-3' 5'-TACAGGATCCACGCGTAGACATTCTGATGACCCAGTCT-3' Lambda chain constant domain MLC.F.Not Lambda chain variable domain MLV.B.Mlu 5'-TGACAAGCTTGCGGCCGCGGTGAGTGTGGGAGTGGACTT GGGCTG-3' 5'-TACAGGATCCACGCGTACAGGCTGTTGTGACTCAGGAA-3' aUnderlined, Recognition sequences of the restriction endonucleases used for cloning into pOPE101 or pSEX81plasmids 453 25/Toleikis/445-458/F 453 09/26/2003, 4:48 PM 454 Toleikis, Broders, and Dübel 11 Place the PCR tubes in a thermal cycler (see Note 5) Denature at 95°C for (see Note 6) 12 Perform 20 cycles with the following cycling program: 45 s of denaturation at 94°C, 45 s of annealing at the appropriate primer hybridization temperature (see Note 9), and 90 s of extension at 72°C After 20 cycles, perform a final extension step for at 72°C 13 Take out 1/10 volume of each PCR reaction for analytical agarose gel electrophoresis on a 2% agarose gel Purify the PCR products using the kit Elute DNA in H2O (see Note 8) 3.2 Cloning into an Expression Vector (pOPE101) Cloning of the two V-region gene fragments is done in two subsequent steps First, the purified PCR product of the light chain is cloned into pOPE101: Digest both the PCR product of the light chain and pOPE101-215(Yol) with MluI and NotI (see Note 10) To prevent self-ligation, the vector should be treated with CIP Run the digests on an agarose gel and gel-purify the vector backbone from the original VL and gel- or PCR-purify the digested light chain Elute plasmid DNA in H2O Estimate DNA concentrations of vector and insert from an agarose gel using a calibrated marker Prepare the ligation mix with an approximate molar ratio vector:insert of 1:3 in a reaction volume of 20 µL The use of 100 ng of total DNA is recommended (see Note 11) Incubate overnight at 16°C Add H2O to a total volume of 50 µL and mix with 10 vol n-butanol, vortex, and centrifuge for 20 at 15,000g at room temperature to precipitate the DNA Let dry and redissolve the DNA in 20 µL H2O Transform E coli XL1-Blue cells (or any other suitable strain) and plate on LB agar plates containing 100 mM glucose and 100 µg/mL ampicillin (LBGA) Incubate overnight at 37°C (see Note 12) Pick five single colonies and grow in mL LB medium containing 100 mM glucose and 100 µg/mL ampicillin Shake overnight at 230 rpm, 37°C Miniprep the plasmid DNA and make glycerol stocks To confirm the presence of the appropriate insert, an analytical digest (run on agarose gel electrophoresis) with MluI and NotI as well as sequencing should be performed In a second step, the heavy chain is cloned: 10 Digest the PCR product of the heavy chain and the ligation product from step with NcoI and HindIII The vector should be CIP-treated 11 From here you can follow steps 2–8 as given for the light chain DNA cloning 12 Confirm the presence of the correct insert by digesting the construct with NcoI and HindIII as well as by sequencing 3.3 Small-Scale Expression Test To confirm the expression and correct size of the scFv fragment, a small-scale expression followed by Western blotting should be performed (see Note 13) Prepare an overnight culture of E coli cells transformed with the appropriate pOPE vector construct in mL of LBGA medium (see Note 14) Dilute 300 µL of the overnight culture into mL (1/20) of LBGA and shake at 37°C and 230 rpm to an OD600 of 0.6–0.8 (see Note 15) Separate the culture into two equal aliquots To one of them add IPTG to a final concentration of 50 µM (see Note 16) 25/Toleikis/445-458/F 454 09/26/2003, 4:48 PM Cloning scFv from Hybridoma Cells 455 Incubate the induced and the control culture for h with vigorous shaking at 270– 280 rpm at 25°C (see Note 17) Incubate the cultures for 10 on ice To check the production of the antibody fragment, take out mL of each culture for immunoblotting Centrifuge at 5000g for at 4°C and resuspend the pellet in 100 µL 2X SDS sample buffer Boil at 95°C for and spin down for at 12,000g before loading the supernatant onto the SDS gel (see Note 18) In addition, a periplasmic fraction can be prepared from the induced culture to test directly for antigen binding affinity in ELISA (see Note 19) Notes Use the RNeasy Mini Kit protocol for the isolation of total RNA from animal cells Use QIAshredder column for the homogenization of the cells Use the Oligotex mRNA Spin-Column protocol for the isolation of poly A+ mRNA from total RNA Use up to 250 µg total RNA for the “miniprep” protocol The concentration and purity of poly A+ mRNA can be determined by measuring the absorbance at 260 nm and 280 nm in a spectrophotometer Use the protocol for First-Strand Synthesis Using Oligo(dT) for the isolated mRNA instead of total RNA, as given The PCR setup with the proofreading ProofStart DNA polymerase can be done at room temperature For detailed information, see the ProofStart™ PCR Handbook Alternatively, it is possible to use other proofreading polymerases, e.g., the Expand High Fidelity PCR System (Roche) Only a few primer combinations will give a PCR product Please note that due to the conditions of the reaction, the same primary sequence may be amplified with several different primer combinations, depending on the individual sequence The size of the resulting PCR product is approx 350 bp If using a thermal cycler with a heated lid, not use mineral oil Otherwise, overlay the reaction with approx 50 µL mineral oil to prevent evaporation The ProofStart DNA Polymerase is activated by this initial heating step For the oligonucleotide primers described in Table 1, an annealing temperature of 54°C should be tried initially If no PCR products are found, decrease the annealing temperature by steps of degrees Use a PCR Purification Kit If multiple bands are found on the gel per reaction, use a Gel Extraction Kit for purification of the correct PCR product (size of approx 350 bp) An annealing temperature of 56°C is recommended 10 Per µg of vector, use U NotI and U MluI, h, 37°C Per µg of insert, apply 30 U NotI and 30 U MluI, h, 37°C 11 A critical factor for the proper function of the T4-DNA ligase is ATP It is recommended to prepare aliquots of 10X reaction buffer, which has been supplemented with ATP to 100 mM and store frozen until used Caution: This (high) ATP concentration inhibits blunt-end ligation reactions 12 Electroporation is recommended because of its unsurpassed transformation efficiency Use half of the ligation and 3–5 µL of electrocompetent cells (Stratagene) and bring to a volume of 50 µL with H2O For a 2-mm-diameter cuvet, use 2.5 kV/25 µF at 200 Ω Alternatively, chemical transformation using a heat shock can be performed 13 To save time, this test can be combined with the generation of cells for DNA “minipreps” and preparation of glycerol stocks by using the remainder of the overnight starter culture 25/Toleikis/445-458/F 455 09/26/2003, 4:48 PM 456 Toleikis, Broders, and Dübel 14 If possible, use glycerol stocks for the preparation of overnight cultures because clones on agar plates can mutate easily after prolonged storage even at 4°C Glucose must always be present in the bacterial growth medium since it is necessary for the tight suppression of the synthetic promotor of the pOPE vector family and thus for the genetic stability of the insert 15 Protein production from pOPE101 cannot be induced in bacteria grown to stationary phase 16 With pOPE-vectors in E coli XL1-Blue, we achieved optimal protein secretion with 20 µM IPTG at 25°C This optimal IPTG concentration can vary between different Fv sequences by a factor of about Higher IPTG concentrations lead to dramatically increased amounts of total recombinant protein, but in this case most of the scFv fragments still carry the bacterial leader sequence and form aggregates However, for immunoblot analysis of total cellular SDS extracts, it is not necessary to discriminate between unprocessed and processed protein Therefore, a higher IPTG concentration is used simply to increase the intensity of the protein band on the blot To optimize expression conditions, IPTG concentrations between 10 and 100 µM should be tested for each individual fusion protein Be aware that IPTG is light-sensitive and may decay during prolonged storage Use stocks aliquoted and stored in brown 1.5-mL tubes at –20°C for not longer than mo 17 A maximum of functional scFv fragments was achieved at 25°C Incubation times longer than h lead to a slight increase in the amount of secreted protein, but a significantly higher contaminant concentration as well, possibly due to increased cell death However, depending on the hybridoma antibody sequence, differences were found in solubility and the ability to be secreted The expression of some antibodies even led to a strong growth inhibition during induction 18 For detection of scFv produced from pOPE101, the monoclonal antibody Myc1-9E10 recognizing the c-myc tag or an antibody to the his tag is recommended HRP-conjugated antibodies to mouse immunglobulins should be applied before TMB-stabilized substrate for HRP (Progen, Heidelberg, Germany) can be used for the detection of bound enzymatic activity 19 Soluble antibody fragments can be isolated from induced E coli cultures by osmotic shock For immunodetection in ELISA, the same antibodies as in Note 18 are suitable Protocols for both methods can be found in Schmiedl et al (24) Acknowledgment We gratefully acknowledge the funding of O.B by the 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(2001) Antibody Engineering Springer-Verlag, New York (ISBN 3-540-41354-5) 26 Sambrook, J., Fritsch, E F., and Maniatis, T (1989) Molecular Cloning A Laboratory Manual, 2nd ed Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 27 Zhou, H., Fisher, R J., and Papas, T S (1994) Optimization of primer sequences for mouse scFv repertoire display library construction Nucleic Acids Res 22, 888–889 25/Toleikis/445-458/F 458 09/26/2003, 4:48 PM ... seriously restricts the size of the sample, and hence the protein antigen has to be From: Methods in Molecular Medicine, vol 94: Molecular Diagnosis of Infectious Diseases, 2/e Edited by: J Decker... result, a variety of differential screening methods have been developed over the last few years (1,2) From: Methods in Molecular Medicine, vol 94: Molecular Diagnosis of Infectious Diseases, 2/e... nitrocellulose membranes was an important milestone for From: Methods in Molecular Medicine, vol 94: Molecular Diagnosis of Infectious Diseases, 2/e Edited by: J Decker and U Reischl © Humana Press Inc.,