UNI SCI C8116 Antibodies as immunochemical tools Spring semester 2025 Hans Gorris Department of Biochemistry March 4th, 2025 Large diversity in the recognition of antigens BCR and antibodies: gene rearrangement + somatic hypermutation => Each individual can recognize any hapten/epitop (linear and conformational epitopes) TCR: gene rearrangement => Each individual can recognize any linear peptide in context with MHC molecule MHC: no gene rearrangement but 3 genes and several thousand alleles in a population => Can bind a large variety of peptides (but not all) => a whole population is well protected but there is an individual risk of missing some pathogenic peptides => populations with a large gene pool are more resistant to an epidemic 2 Interaction of TCR with a peptide on MHC class I a chain of (A) (B) class I MHC protein => Only linear peptide epitopes Summary of interplay between TH and B cells Antigen T cell epitope (binds to MHC, recognized byTCR) B cell epitope (binds to BCR) DC acquires antigen and moves to lymph node DC processes and presents antigen to CD4 Tcell Antigen — Dendritic cell (DC) Taj Efl T cell interacts with B cell through TCR-MHC interaction jjj T cell interacts with B cell through TCR-MHC interaction and by CD40L-CD40 interaction-* IL-4 B cell activated and secretes IgM -► B cell acquires antigen B cell processes antigen and displays class II MHC-peptide complexes on surface CD40L CD40 Hypermutation Class switch recombination Other isotypes High affinity immunoglobulin y= Antibodies as immunochemical tools The "tools": Immunology antibodies Immunoassay effector B cell (plasma cell) |_| 1 fi,m 5 Antigenic determinants: hapten Immunization generates antibodies only against large molecules, e.g. proteins Antibodies against small molecules (haptens) must be produced by coupling (typically derivatized) small molecule onto the surface a large carrier protein. . lysine 0=C amino acid I H-C-CH2-CH2-CH2-CH2- ■ N-HB polypeptide backbone of protein Carrier NH -^^-N02 N02 dinitrophenyl group (DNP) Hapten Other examples: toxins, pharmaceticals hormones... Definition of hapten: A low-molecular weight molecule which contains an antigenic determinant but which is not itself antigenic unless bound to an antigenic carrier => Why do we need a carrier protein to launch an immune response against DNP? - Antigenic determinants: hapten Immunogens are always antigens but not all antigens are immunogens hapten 1MMUNOGEN spacer / & carrier/ Protein (e.g. KLH) SPACER BINDING EFFECT Antibody binding site carrier / Protein (e.g. KLH) Cross reactivity (CR) in competitive assay DCF 5-OH-DCF 4'-OH-DCF DCF-GLU Ibuprofen Ketoprofen Meclofenamic Fenoprofen Mefenamic Tolfenamic acl Some definitions Antibodies, or immunoglobulins (Igs), are y-globulin proteins folded into well defined three-dimensional structures synthesized by living organisms, e.g. mice, rabbits or goats, or by living cells, in response to the presence of a foreign substance known as the antigen. Immunogen: Molecule that is capable of eliciting an immune response by the immune system of an organism. Antigen: Molecule that is able to bind to the product of that immune response: the antibody. Epitope: An epitope is a specific location on the surface of an antigen that has a particular molecular structure and that is recognized by a particular antibody or a set of specific antibodies that the epitope elicits during the immune response. Hapten: Small molecules (< 5000 Dalton) that need to be conjugated to a carrier protein (e.g. bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH) or ovalbumin) to elicit the immune response. Polyclonal vs. monoclonal antibodies polyclonal monoclonal Antibodies that are collected from sera of exposed animal Individual B cell hybridoma is cloned and cultured. Secreted antibodies are collected from culture media recognize multiple antigenic sites of injected substance recognize ONE antigenic site of injected substance Polyclonal antibodies (Pab, antiserum) - Antibody production: quick and inexpensive - Immunization procedure: • animal is immunized with pure antigen (immunogen) and with adjuvant (substance that strengthens the immune response) • the immunization is repeated (boost) • the animal's blood is collected - Antibody host species: • typically mouse/rabbit • sheep/goat in case large antibody amounts are needed (for commercial use 11 Polyclonal antibodies + and + Fast preparation Inexpensive Sometimes very high affinity which is difficult to obtain with monoclonal antibodies (e.g. anti-steroid antibodies) May be advantageous for the detection of very heterogenous antigens Pros Greater reagent versatility Limited amounts (typically not sufficient for in excess reagent systems) Variations between batches Often lack full antigen specificity Cannot discriminate between closely related antigens Pure antigen required for immunization Cons Potential for reduced specificity Shorter development; typically available first High lot-to-lot variability 12 Generation of monoclonal antibodies => first described by Köhler/Milstein (1975) mouse immunized with antigen X I cell making anti-X antibody B lymphocytes (die after a few days in culture) mutant cell line derived from a tumor of B lymphocytes (myeloma cells) (cells grow indefinitely in normal medium but die in selective medium) FUSION (PEG or electrofusion) i resulting hybridoma cells cultured in multiple wells 13 Generation of monoclonal antibodies only hybridoma cells survive and proliferate in the selective medium (HAT) secreted anti-X antibody supernatant tested for anti-X antibody, and cells from positive well cultured at ~1 cell per well 1 \ \ 1 I I \ \ 1 • > • 0 • > > • > limiting dilution cells allowed to multiply, and individual supernatants tested for anti-X antibodies positive clones provide a continuing source of /O o anti-X antibody /o°ooN 14 Generation of monoclonal antibodies 1. Hyperimmunize mouse with antigen and adjuvans (immunostimulant) 2. Fuse B cells with tumor (myeloma) cell line in PEG (polyethylene glycol) or by electrofusion 3. Limiting dilution in 96 well MTPs to fractionate fused cells in HAT medium (hypoxanthine, aminopterin, thymidine) HAT Selection Genotype:* Cell type: tk immortal HAT-sensitive plasmacytoma tk+/tk- fused ^ m hybrid ^ tk + mortal plenic B-c HAT fate: Explanation: DIES Unable to synthesize DNA: (1) Thymidine kinase* mutation causes a loss-of-function in the "salvage" pathway and (2) Aminopterin blocks "De novo" pathway. SURVIVES Immortal and restored DNA synthesis: (1) Immortality from plasmacytoma and (2) rescued ability to synthesize DNA due to restored thymidine kinase* function. DIES Mortal: (1) Functional DNA synthesis, but (2) eventually dies because of limited number of replication cycles 15 *HGPRT (hypoxanthine-guanine phosphoribosyltransferase) mutants can be used in place of TK (thymidine kinase) mutants Expand in mice or in vitro Freeze Expand and Clone Positive Wells I Assay Antibody Freeze-*— Redone Positives Monoclonal Antibody Production In Vitro Culture (10 ug/ml mAb) in vitro material is less concentrated and contains bovine serum Ascites Tumor (5 mg/ml mAb) ascites fluid contains high [mAb] and has some contamination with the mouse natural Ig 16 Monoclonal antibodies + and Constant supply of same antibody (from in vitro culture) Constant affinity and specificity IgG fraction yields in practice a « 100 % active preparation 100 percent epitope specificity => possible to design very specific assays for closely related antigens, and I posttranslational variants (fragments, I cleaved forms, sugar variants etc.) I No need for 100 % purity of Ag for I immunization Sometimes too specific (does not recognize a genetic or other variant) Often of lower affinity than polyclonals => especially important if used in a competitive assay (in sandwich assay excess compensate the lower affinity) High specificity Constant supply 17 Monoclonal antibodies can be too specific if there is a common genetic variant of protein -> false negative result! 18 Using antibodies as immunochemical tools Polyclonal vs. monoclonal antibodies as a reagent An antibody reagent differs in the way how it is produced against the analyte. The production determines the recognition specificity for analyte epitopes*. Polyclonal and monoclonal antibodies are very similar protein reagents except for the amino acids in the paratope region. *ln the context of antibodies, we only talk about B cell epitopes! 19 Handling of antibodies (IgG) Advantages as chemical reagents: - well soluble (unlike IgM) - also active with low salt content - binding over wide pH range (pH 4-9.5) Storage: - can be stored in sterile serum for several months up to a few years at 4 ° C - long-time storage after snap freezing in liquid nitrogen at -20° C or better at -80° C - freeze drying (lyophilization): mainly from commercial suppliers Problems: - damage through bacterial growth => add 0.02% (final concentration) of sodium azide (NaN3) or 0.01% thimerosal - isolated, purified antibodies are prone to aggregation after freezing and at low concentrations lead to losses by attachment to plastic surfaces => add 1% bovine serum albumin (BSA) - the freezer should not have a de-frosting cycle! - avoid repeated thawing / freezing; better prepare small aliquots - some antibody-enzyme conjugates (e.g. horseradish peroxidase) lose activity after freezing => dilute with glycerol (50%) and store at -20° C (sample does not freeze) 20 Labeling of antibodies with fluorescent dyes Structure Ft Name Amax (nm) Reversible unbinding of antibody-antigen complexes Acidic conditions Optimal: pH 2.6; with very high affinity antibodies harsher conditions are required pH 1.8 at 4° C for a short time, but leads to some damage. Alkaline conditions Optimal: pH 11.2; harsher conditions damage the antibody even more strongly than acidic conditions. Chaotropic ions CI, I", Br, SCN", typical eluents: 3 M MgCI2, 1-3 M NaSCN. Epitopes Higher concentration of competing free antigen, hapten, synthetic peptides Elevated temperatures Not in use any more 22 Choosing your antibody host species We discussed the following animals for obtaining antibodies: - Mice: easy breeding, but only small amounts - Rabbits: larger amounts => for polyclonal antibodies - Goats: large amounts => for polyclonal antibodies Other consideration for using different species: - it is not possible to obtain anti-mouse IgG by immunizing mice (=> immunol. tolerance) - if a mouse sample is to be investigated, mouse-antibodies may show cross-reactivity. secondary antibody => anti-mouse IgG direct Similar considerations for blocking reagents: - blocking reagents should not be obtained from species of the primary antibody because then all non-specifically bound primary abs would also be detected (in addition of non-specific binding of secondary ab) indirect detection 23 Antibodies as immunochemical reagents => Antibodies are used as bioanalytical reagents to specifically detect and quantify other molecules Antigens Epitope Antigen Antigen-binding site paratope Antibody 24 Continuous vs. discontinuous eptiopes Continuous epitope: short peptide or denatured protein structure, epitope consist of sequential amino acids peptide chain antibody bindi Discontinuous epitope: present only in 3-dimensional protein structure, epitope comprises non-sequential amino acids peptide chain nition site 25 Excursion: Epitope mapping How do we know to what epitope an antibody binds? o o chromogen °o°o° (BCIG) Epitope mapping *. . ■ * • » * «i til "fe- 4r ft * -f c? ^» lo f: I.. CMV2 6-decapeptide scan 10 20 30 40 50 IEGRGKSRGGGGGGGSLSSLANAGGLHDDGPGLDNDLMNEPMGLGGLGGGGGGGGKKH 1.IEGRGKSRGG 2.EGRGKSRGGG 3.GRGKSRGGGG 4.RGKSRGGGGG 5.GKSRGGGGGG 6.KSRGGGGGGG 7.SRGGGGGGGS 8.RGGGGGGGSL 9. GGGGGGGSLS 10. GGGGGGSLSS 11.GGGGGSLSSL 12.GGGGSLSSLA 13.GGGSLSSLAN 14.GGSLSSLANA 15.GSLSSLANAG 16.SLSSLANAGG 21.ANAGGLHDDG 2 2.NAGGLHDDGP 23-AGGLHDDGPG 2 4.GGLHDDGPGL 2 5.GLHDDGPGLD 26.LHDDGPGLDN 27.HDDGPGLDND 28.DDGPGLDNDL 29.DGPGLDNDLM 3 0.GPGLDNDLMN 31.PGLDHDLMNE 32.GLDNDLMNEP 33.LDNDLMNEPM 34.DNDLHNEPNG 41.PMGLGGLGGG 42.MGLGGLGGGG 4 3.GLGGLGGGGG 4 4.LGGLGGGGGG 4 5.GGLGGGGGGG 4 6.GLGGGGGGGG 4 7.LGGGGGGGGK 48.GGGGGGGGKK 49.GGGGGGGKKH 17.LSSLANAGGL 18.SSLANAGGLH 19.SLANAGGLHD 2 0.LANAGGLHDD 3 5.NDLMNEPMGL 36.DLMNEPMGLG 3 7.LHNEPMGLGG 38.MNEPMGLGGL 3 9.NEPHGLGGLG 40.EPMGLGGLGG => But continuous epitopes only 27 Overlapping eptiopes Even small analytes can have multiple epitopes, but antibody binding to one epitope blocks another epitope, i.e. these epitopes are overlapping epitope-1 epitope-2 short peptide antibody-1 antibody-2 28 Non-overlapping eptiopes a folded protein "the analyte" antibody-1 Monoclonal antibody reagent all antibodies are from the same B cell clone => reagent consist of identical antibodies, and all recognize and are specific for only one identical epitope identical paratopes .. will bind only to one specific epitope in the analyte - unless there are multiple identical epitopes in the same analyte epitope-1 * epitope-2 Polyclonal antibody reagent Mix of different B cell clones ... will bind simultaneously to => reagent consist of antibodies that have one or several non-overlapping different paratopes and recognize different epitopes in the analyte Antibody affinity 32 Affinity of an antibody Tight fit / Less interaction Little interaction / high affinity / lower affinity very low affinity 33 Antibody-antigen binding reaction Ab (antibody) Ag (antigen/analyte) AgAb + Ag + Ab 5=^ AgAb 34 Surface plasmon resonance (SPR) incident prism, or grating light gold film r (~50 nm) L reflected-light detector antibody attached to gold film surface plasmons excited in gold film by light at a specific resonance angle ] plasmon-induced evanescent electric field extends beyond gold film solution of antigen molecules 35 Determining the affinity of antibodies by SPR (1) Binding of antigen to surface immobilized antibodies increases the refractive index of the surface layer. (2) The resulting change of the resonance angel for plamson induction can be measured by a photodetector. solution of antigen molecules association buffer solution dissociation 10 min antigen added buffer wash But caution: SPR measures association (on) and dissociation (off) rate constants when one binding partner is surface-bound. Affinity constants, however, are defined for both binding partners free in solution! 36 Affinity of an antibody Ag + Ab AgAb ka[Ag][Ab] = kd[AgAb] reaction velocities at equilibrium: Bound antibody k = — = lr [A AW\ DUUIIU dMUL Ka _ lAgAD] < and antjgen kd [Ag] [Ab] free antigen free antibody ka: association rate constant (on rate) kd: dissociation rate constant (off rate) K: affinity constant 37 Affinity of an antibody 120 r finedictable from affinity MAb 1 MAi) :■ + extremely stable binding: off-rate is very slow poorly suited for immunoassay due to dissociation problems, e.g. during wash or during 2nd step if used as capture antibody 601 Fig. 1.6 Response curves illustrating the interaction of P24 antigen (125 nM) with three different monoclonal antibodies (MAbs). v/ Sensorgrams for 3 moncolonal antibodies against HIV p24 surface Ag 38 Affinity of an antibody k [AnAh} *_Bound antibody „ _ _ \.niJ^u\ and antigen ~kd~[Ag][Ab] free antigen free antibody approximate calculation of concentrations in equilibrium: if [Ag\toi « [Ab]to\> on|y a very small antibody fraction is present in the complex [AgAb] => [Ab] * [Ab]tot [Ag]tot =/QV lA9A® \A n A 111 = ^toM^tot K [Ab]M =^Ab]} [AgAb] IW1 ([Ab]tot K) + l \ free (unbound) concentrations 39 Affinity of an antibody Calculating the equilibrium concentration [Ab]tot[Ag]tot K [AgAb] = ([Ab]tot K) + 1 [Ab]m = 1 * 10"9 M [Ag]M = 1 * 10"12 M (i.e. much smaller) K= 1 * 109 M-1 by calculating we get [AgAb] = 0.5 * 1012 M (i.e. 50%) "rule of thumb": when [Ab]M= 1/K then [AgAb] = 50% [Ag]tot [Ab]ta= 10/K then [AgAb] = 90% [Ag]tot [Ab]M= 0.1/K then[^d] = 10%[Ag]tot 40 Affinity of an antibody Antigen per tube (pg) Figure 8.4 Estimation of filled antibody sites, at different concentrations of antigen, for three antibodies with different affinity constant (1/mol). ■ = 1 x 109, • = 1 x 1010, A = 1 x 10n. Affinity of an antibody: Scatchard plot linearization: [AbAg] bound Ag K ([Ab]tot - [AbAg]) = ([Agltot" [AbAg]) free Ag Affinity of an antibody: Scatchard plot B => Typcially replaced by non-linear fitting using computer programs 43 Antibody engineering 44 Excursion: Antibody enginering for therapy (A) Fab (C) Fc CH2 CH3 Murine (-momab) Humanized (-zumab) (B) Chimeric (-ximab) (D) Human (-umab) Natural antibodies (raised in mice) are potentially immunogenic => Potential side effects 45 Therapeutic antibodies (market value) 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 Caplacizumab (Cablivi): anti-vWF for acquired aTTP Romosozumab (Evenity): anti-Sclerostin for osteoporosis Rituximab (Rituxan): anti-CD20 for Non-Hodgkin lymphoma Daclizumab (Zinbryta): an1i-CD25 for multiple sclerosis Abciximab (Reopro): anti-GP llb/llla for prevention of blood clots in angioplasty Muromonab-CD3 (Orthoclone OKT3): anti-CD3 tor kidney transplantation rejection treatment Successful test of anti-lymphoma Ab First mAb Burosumab (Crysvita): anti-FGF23 for XLH Ibalizumab (Trogarzo): anti-CD4for HIV infection Erenumab (Aimovig): anti-CGRPR for migraine prevention Atezolizumab (Tecentriq): anti-PD-L1 for bladder cancer Reslizumab (Cinqaero, Cinqair): anti-IL-5 for asthma Trastuzumab (Herceptin): anti-HER2 for BC Infliximab (Remicade): anti-TNFa for Crohn's disease 5.4 Pembrolizumab (Keytruda): anti-PD-1 tor melanoma Nivolumab (Opdivo): anti-PD-1 for melanoma & NSCLC Ramucirumab (Cyramza): anti-VEGFR2 for GC Ipllimumab (Yervoy): anti-CTLA-4 for metastatic melanoma Brentuximab vedotin (Adcetris): anti-CD30 for Hodgkin lymphoma, systemic ALCL Ustekinumab (Stelara): anti-IL-12/23 for Psoriasis Panitumumab (Vectibix): anti-EGFRtorCRC Ranlbizumab (Lucentis): 43 anti-VEGF-A for Macular degeneration 86 300 Billion In 2025 A I f O150 115 98 75 78 50 92 Avelumab (Bavencio): anti-PD-L1 for MCC Durvalumab (Imfinzi): anti-PD-L1 for bladder cancer Brodalumab (Siliq, Lumicef): anti-IL-17R tor plaque psoriasis Necitumumab (Portrazza): anti-EGFR for NSCLC Dinutuximab (Unituxin): anti-GD2 for neuroblastoma Pertuzumab (Perjeta): anti-HER2 for BC 45 26 Denosumab (Prolia): anti-RANKL for bone loss 35 Eculizumab (Soliris): anti-C5 for Paroxysmal nocturnal hemoglobinuria 11 Bevacizumab (Avastin): anti-VEGF for CRC; Cetuximab (Erbitux): anti-EGFR for CRC Omalizumab (Xolair): anti-lgE for asthma 0.3 Adalimumab (Humira anti-TNFa for RA x* Q> C§> ^ rSp cSť cST rSF rS> & ^ ^ $P jS* ft«3 «CS3 cí^ cvNN cv^ cí^ ^ ^ ^ cvN^ ^ čí?* TT ^ ^ n5> n5> n5» n5> nS> f Year 46 Humanized antibodies No. Drug Indication (1st US FDA Approval Year) Company 2018 Revenue (USD, billion) 1 Adalimumab Rheumatoid arthritis (2002) AbbVie $19.9 bn (Humira) Psoriatic arthritis (2005) Ankylosing spondylitis (2006) Juvenile Idiopathic Arthitis (2008) Psoriasis (2008) Crohn's disease (2010) Ulcerative colitis (2012) Hidradenitis suppurativa (2015) Uveitis (2018) 2Nivolumab (Opdivo) Melanoma (2015) Non-small cell lung cancer (2015) Renal cell carcinoma (2015) Head and neck squamous cell (2016) Bristol-Myers Squibb $7.6 bn 3Pembrolizumab Melanoma (2014) Merck & Co $7.2 bn (Keytruda) Head and neck cancer (2016) Non-small cell lung caccer (2015) Lymphoma (2018) Cervical cancer (2018) Microsatellite instability-high cancer (2018) 4Trastuzumab Breast cancer (1998) Roche $7.0 bn (Herceptin) Gastric cancer (2010) 5Bevacizumab Colorectal cancer (2004) Roche $6.8 bn (Avastin) Non-small cell lung caccer (2006) Breast ERB2 negative cancer (2008) Renal cell carcinoma (2009) Glioblastoma (2011) 6Rituximab, Non-Hodgkin's lymphoma (1997) Roche $6.8 bn 47 Recombinant antibody fragments Artificial smaller constructs Natural IgG antibody Paratope Disadvantage of antibodies as a reagent: relatively large size Flexible peptide linker qpCw (Single-chain OUT V variable fragment) => same paratope, but much smaller 48 Recombinant antibody fragments Immortalization of hybridomas through cloning or generation of new antibodies without immunization • Greater speed of production (E. coli batch fermentation) • New specificities especially for poor immunogens • Possibility to fine-tune antibody specificity and affinity • Possibility to tailor make the antibody to perform special tasks • tags, handles (for conjugation, immobilization) • fusing to other protein (e.g. enzymes) Likely to be increasingly used in miniaturised systems to enable full control of antibody performance. 49 Heavy chain antibodies Shark HcAb Front. Immunol., 2017 https://doi.org/10.3389/fimmu.2017.00977 From heavy chain antibodies to nanobodies heavy-chain antibody IgG antibody Nanobodies: Detection of hidden epitopes Advantages of nanobodies - Mass: ca. 15 kDa (IgG: 150 kDa), 2.5 nm diameter (IgG 15 nm) - High solubility - Rapid targeting and fast blood clearance - Detection of "hidden" epitopes - Easy cloning: Recombinant engineering and protein expression in vitro in bacterial production systems are much simpler - Very stable and heat resistant (no cold storage required) - Simple genetic structure allows easy re-engineering of nanobodies to introduce new antigen-binding characteristics or attach labels 53 Recombinant nanobodies fluorescent chromogenic chemical protein enzyme dye a Chromobodies. b Detection of the nuclear lamina with lamin chromobody in living cells. Confocal images of HeLa cells coexpressing lamin chromobody (green) and red fluorescent histone H2B as a mitosis marker. Scale bar: 10 urn ^ 54 Anal. Bioanal. Chem. (2010) 397: 3203-3208 Phage display using filamentous phage M13 virus DNA • Infects / replicates in E. coli • Protein coat: major coat protein: pVIII minor coat proteins: pill, pVI, pVII, pIX • The phage can be engineered to display foreign peptides or proteins as a fusion with one of the coat proteins, most commonly pill. • The genomic DNA encoding for the coat proteins is enclosed within the protein coat. => Each protein remains connected to its encoding DNA Displayed protein George Smith / Greg Winter: Nobel prize in chemistry 2018 55 Construction of phage displayed protein libraries Protein engineering by in vitro evolution Selection cycle Identification of high affinity Option to introduce random mutations (e.g. error-prone PCR) Single-domain antibody (nanobody) Heavy chain antibodies Conventional antibodies Ag Peripheral blood lymphocytes RNA extraction and reverse transcription VHH CH2 CH3 VHH 1 Ü VHH pill phagemid vector V / / ü Amplification of variable regions from cDNA Purification and digestion of amplicons Cloning in a phagemid vector and library preparation VH CH1 CH2 CH3 I - VL CL Uvh 0vl scFv VL VH pill phagemid vector Phage display library / 58 Production of recombinant antibodies Expression system (Gene of Ab fragment known) mRNA isolation, cDNA synthesis and gene synthesis f f lgK \ lgH-G1 or k J TransfectiontoHEK293E ©A ^ . * ___ Recombinant antibody purification Functionality assessment I I o E O "5 Phage display (Gene of Ab fragment unknown) Phage library displaying 1111 ni 11: ■ 11 1 !■■ ■ . i. ■: i: i . 11 ■ Ani body Hagmerrt (schv) - Next panning cycle Washing ' in bacteria fllS M Äji ÄÄ ífň (Si: Nucleus Mammalian Cell qqocgoBccoBo IgG AKpreuion VKtOf Screening for specificity scfv Cloning Fc- *=* „ *=* Purification v secretion IB i-- •• 59 Alternatives for antibodies 60 Aptamers Complementatary base pairing Binding through: (1) 3-dimensional, shape-dependent interactions (2) hydrophobic interactions, base-stacking, intercalation 61 SELEX* single-stranded DNA library 5'-Primer random DNA 3'-Primer i-' ' —i i-i i —i 2 in15 S ~10 transcription dsDNA t PCR\ cloning & ^sequencing uO o Aptamer cDNA reverse transcription *systematic evolution of ligands by exponential enrichment => in vitro selection target immobilized on microsphere 3 C □ 0<$ selected RNA unbound RNA 62 SELEX* Step 1: Bind oligonucleotide Step 2: Elute oligonucleotides Step 3: Perform PCR Step 4: Repeat steps 1 library and discard non-binder that bind desired targets to amplify eluted binders through 3 using enriched 63 Aptamers: Assay designs 64 Molecularly imprinted polymer (MIP) "Plastic antibodies" binding polymerization template elution 65