MUNI SCI C8116 Immunochemical techniques Immune system, part II Antibodies as immunochemical tools Spring semester 2024 Hans Gorris Department of Biochemistry February 27th, 2024 Topics of the lecture Part A: The immune system and its sharpest weapon: antibodies Part B: Antibodies as immunological tools Part C: Immunoassays Part D: Immunoaffinity and other protein-protein affinity techniques Part E: Advanced fluorescence microscopy for (life) cell imaging Structure of IgG Two identical antigen binding sites antigen-binding site antigen-binding site light chain heavy chain heavy chain LIGHT CHAIN variable constant region region (k type or \ type) r H2N- 1 .-COOH HEAVY CHAIN H2N-k variable region constant region (a, 8, e, 7, or |x type) 2 heavy (50 kDa, dark green) 2 light chains (25 kDa, light green) total mass: 150 kDa l-COOH J hooc cooh 3 Clonal selection theory precursor cell different resting B cells □ antigen PROLIFERATION AND DIVERSIFICATION IN BONE MARROW ANTIGEN BINDING TO SPECIFIC B CELL (Bp) IN PERIPHERAL LYMPHOID ORGAN PROLIFERATION (CLONAL EXPANSION) AND DIFFERENTIATION OF Bß CELLS antibody-secreting effective B(3 cells secreted antibodies There's only a single antibody specificity per B cell => mass production Antibody classes fgA (dimer) IgM (pentamer) Y dwin £ chdin Heavy chain defines the class of antibody Same light chain: k or A PROPERTIES CLASS OF ANTIBODY IgD IgG IgA Heavy chains 8 7 a e Light chains K or X K or X Kor X Kor X KorX Number of four-chain units 5 1 1 1 or 2 1 Percentage of total Ig in blood 10 <1 75 15 <1 Activates complement ++++ - ++ - - Crosses placenta - - + - - Binds to macrophages and neutrophils - - + - - Binds to mast cells and basophils => B cells can switch between the production of antibody classes primary secondary classes of antibody T T cells and T cell receptor (TCR) effector T cel I 1 |xm binding site i-1 a chain H2N nh2 ß chain EXTRACELLULAR SPACE plasma membrane cooh cooh CYTOSOL 6 B and T cell maturation follow a similar course Stem cell Pro-lymphocyte Pre-lymphocyte Pre-BCR B lineage T lineage Immature lymphocyte BCR Mature lymphocyte Differentiatec effector lymphocyte Pre-TCR TCR Cyt 7 / okines r Bone marrow or thymus Periphery No Self antigen Foreign antigen r- i * Pre-antigen Early maturation ^ ^ . a . . ^ ^ receptor and expansion ^ ^ K expression Completion of antigen receptor; selection of receptor repertoire; differentiation Performance of j effector functions Antigen presentation ACTIVATED T CELLS MIGRATE TO SITE OF INFECTION VIA THE BLOOD 8 5 |xm Antigen-laden dendritic ceH U andmovestofoil.de Soluble antigen Mature T and Bceiis are deliveredI via the circulation and take up residence »n lymph nodes - Afferent lymphatic vessel Antigen Activation of T cell by antigen-laden, activated dendritic cell; activated T cells may re-enter circulation Blood vessels Efferent lymphatic vessel Hj Activated T cells interact with B cells, leading to B-cell differentiation and antibody production Better double check! B cell epitope T cell eptiope recognition Antigen T cell epitope (binds to MHC, recognized byTCR) + B cell epitope (binds to BCR) recognition BCR Two-factor authentication Pick any two: Something you know, something you have, something you are TCR Minimizing the risk of wrong classification (friend/foe) to prevent e.g. autoimmune diseases, allergies 10 T cell receptor (TCR) Generation of TCR diversity (/-chain locus rL1 Va1 L2 Vu2 L3 Va3 LVux70-80 -, Jax61 a-chain 70-80 V gene segments 61 J gene segments => 5000 combinations 'a ß-chain locus ,L1 Vß1 L2 Vß2 L3 Vß3 L Vßx52 -, P-chain 52 V gene segments (2 D gene segments) 13 J gene segments => 700 combinations Dpi Jpi x 6 "p1 Dß2 Jß2X * Cp2 12 Generation of TCR diversity germline DNA recombination rearranged DNA transcription splicing translation translation splicing transcription rearranged DNA recombination germline DNA V„2 V„i a 3 mm => Same enzymes are used for the gene rearrangement as in B cells « Pi => about 3.500.000 combinations Vpi Dpi Jp Cpi Vpn Vpi Dpi Jp Cpi Dp2 Jp Cp2 13 Unlike BCR no somatic hypermutation => only lower affinity (Ka = 105-107 IVM) T cell activation receptor for proteins activated dendritic cell ATCR recognizes the antigen only in context of an MHC 14 Major histocompatibility complex (MHC) CYTOTOXIC T CELL HELPER OR REGULATORY T CELL or target cell Properties of Human Class I and Class II MHC Proteins CLASS 1 CLASS II Genetic loci Chain structure Cell distribution Presents antigen to Source of peptide fragments Polymorphic domains Recognition by co-receptor HLA-A, HLA-B, HLA-C a chain + (^-microglobulin most nucleated cells cytotoxic T cells mainly proteins made in cytoplasm ai+«2 CD8 DP, DQ, DR a chain + p chain dendritic cells, B cells, macrophages, thymus epithelial cells, some others helper T cells, regulatory T cells mainly endocytosed plasma membrane and extracellular proteins «, + P*i 15 CD4 14 Major histocompatibility complex (MHC) Class I MHC protein Class II MHC protein => on (almost) all cells => Only on professional antigen-presenting cells (e.g. dendritic cells) peptide-binding site peptide-binding site 16 14 Peptide bound to MHC peptide-binding groove COOH TOPVIEW CYTOSOL 17 SIDE VIEW hooc ^ Peptide bound in the groove of MHC Peptides bound to MHC-I: Peptides bound to MHC-II: 8-10 amino acids long 10-12 amino acids long 18 Allelic variation in MHC genes 4 1 "3 «1 variability mhc class i variability «1 a, «3 jlIJm!_LjJjLjlJ4 1-T I r T---1-II-1-1-1-1-1— 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 residue Aft P2 /la, mhc class ii variability Pi JUIljuJijlj r~n——1——1-1-1-1-1-1— 0 20 40 60 80 100 1 20 140 1 60 180 200 residue Red: peptide binding regions 19 Human MHC genes class II MHC genes class I MHC genes 1 1 1 1 1 1 ^~ BB Ufa human chromosome 6 ß a ß a ß ß a L HLA complex maternal MHC class II paternal MHC class II maternal MHC class I paternal MHC class I There is a large variability of MHC molecules in a population, but in each individual only: MHC-I 2x HLA-DP 2x HLA-DQ 2x HLA-DR => 6-8 IHC-I 2x HLA-A 2x HLA-B 2x HLA-C =>6 20 Structural comparison: antibody, MHC and TCR 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 22 Interaction of TCR with a peptide on MHC class I a chain of (A) (B) class I MHC protein => Only linear peptide epitopes 23 Friend / foe recognition by T cells precursor cell no TCRs expressed DEATH BY DEFAULT 1 1 DIVERSIFICATION OFT CELL RECEPTORS (TCRs) TCRs with no recognition of self MHC + self peptide DEATH BY DEFAULT apoptotic cell TCRs with strong recognition of self MHC + self peptide TCRs with weak recognition of self MHC + self peptide NEGATIVE SELECTION (signaled death) POSITIVE SELECTION (signaled survival) SURVIVAL and MATURATION to peripheral lymphoid organs 24 T cell recognition of antigens is MHC restricted Tcell a TCR MHCa I antigen-presenting cell Recognition MHC restriction T cell MHCE antigen-presenting cell No recognition T cell antigen-presenting cell No recognition 25 T cell recognizes viral antigen and host target cell virus A strain-X mouse cytotoxic T cells added to strain-X mouse fibroblasts infected with virus B strain-X mouse fibroblasts infected with virus A strain-Y mouse fibroblasts infected with virus A T cells kill target cells NO •: • .TV-: : • YES NO 26 Co-receptors on T cells Serves as a receptor for HI virus binding CD8 protein class 1 MHC protein TCR variable part CD4 protein dendritic cell or target cell invariant part dendritic cell or target cell class II MHC protein 27 Excursion: HI virus infecting T cell HIV 200 nm membrane nucleocapsid chemokine receptor CHEMOKINE RECEPTOR CD4 ATTACHMENT BINDING MEMBRANE INSERTION CONFORMATIONAL FUS|0N CHANGE EXTRACELLULAR SPACE CYTOSOL ENTRY OF VIRAL NUCLEOCAPSID => depletion of TH cells: AIDS (Aquired Immunodeficiency Syndrome) 28 Classification of T cells WBm 1 Cytotoxic T cells (Tc) (CD8+ cells) :> recognize peptides on MHC I effector T eel I T helper cells (TH) (CD4+ cells) => recgonize peptides on MHC II ThO, TH1 infections by microbes TH2 infections by parasites Suppressor T cells regulatory function 29 Topologically equivalent compartments secretory Golgi nucleus vesicle apparatus l lysosome 7 autophagosome Antigen acquisition sites Cytosolic pathogens Intravesicular pathogens Extracellular pathogens and toxins macrophage Degraded in Cytosol Endocytic vesicles (low pH) Endocytic vesicles (low pH) Peptides bind to MHC class 1 MHC class II MHC class II Presented to Effector CD8 T cells Effector CD4 T cells Effector CD4 T cells Effect on presenting cell Cell death Activation to kill intravesicular bacteria and parasites Activation of B cells to secrete Ig to eliminate extracellular bacteria/toxins 31 Antigen presentation by MHC-I plasma membrane VIRUS INFECTION viral RNA internal viral viral envelope ENDOCYTOSIS AND DELIVERY TO ENDOSOME endosome MO assembled class I MHC protein with bound peptide endosome FUSION OF VIRUS WITH ENDOSOME MEMBRANE AND ESCAPE OF VIRAL RNA INTO CYTOSOL RNA A REPLICATION AND TRANSLATION OF VIRAL RNA PROTEOLYSIS OF SOME VIRAL PROTEIN MOLECULES BY PROTEASOMES dendritic cell or target cell internal viral protein \ class I MHC a chain chaperone protein TRANSPORT OF PEPTIDES INTO ER LUMEN BINDING OF PEPTIDE TO a CHAIN STABILIZES ASSEMBLY OF a CHAIN WITH ^MICROGLOBULIN; PEPTIDE-MHC COMPLEX IS THEN TRANSPORTED TO THE GOLGI APPARATUS ^-microglobulin 32 Activation of cytotoxic T cells Virus-infected cell Cytotoxic T cell kills infected cell {&!■ # \ —* L virus infected cell \ J \» cytotoxic \ \ / TCe" killed infected cell 5 |xm centrosome Cytotoxic T cells induce apoptosis A Perforin-dependent killing effector cytotoxic T cell perforin l/JJ molecules serine proteases target cell B Fas-dependent killing effector cytotoxic T cell Fas ligand (trimer) Fas adaptor protein assembled perforin channel inactive procaspase-8_ 111 apoptotic target cell activated caspase-8 apoptotic target cell target cell 34 Activation of a B cell by an antigen and TH cell B cell antigenic T cell antigenic determinant ^determinant BCR endosome protein antigen recognition of peptide-class-ll-MHC complex by antigen-specific helper Tcell effector T cell delivers signals to activate Bcell B cell receptor binds native protein antigen and transfers it to endosome following endocytosis immunological synapse antigen is degraded, and peptide is presented at B cell surface bound to a class IIMHC protein cytokines B cell proliferates and differentiates into an antibody-secreting effector cell co-stimulatory protein 35 Antigen presentation by MHC- helper T cell plasma membrane folded protein antigen early endosome o / late endosome ENDOCYTOSIS AND DELIVERY TO ENDOSOME fragment of invariant chain LIMITED PROTEOLYSIS OF PROTEIN ANTIGEN AND OF INVARIANT CHAIN, LEAVING FRAGMENT OF INVARIANT CHAIN IN BINDING GROOVE OF MHC PROTEIN class II MHC protein INVARIANT CHAIN DIRECTS CLASS II MHC PROTEIN TO LATE ENDOSOME invariant chain Golgi apparatus trans Golgi network dendritic cell HLA-DM PROTEIN CATALYZES RELEASE HLA-DM OF INVARIANT CHAIN FRAGMENT AND BINDING OF ANTIGEN-DERIVED PEPTIDE 11 II DELIVERY OF PEPTIDE-MHC COMPLEX TO PLASMA MEMBRANE FOR RECOGNITION BY HELPER T CELL 4 36 Summary of interplay between TH and B cells Antigen T cell epitope (binds to MHC, recognized byTCR) » B cell epitope (binds to BCR) Antigen — (Vk (9 • DC acquires antigen and moves to lymph node DC processes and presents antigen to CD4 Tcell WlOnJT \ '~ölJ uo J rHelpei Tcell activated Efl T cell interacts with B cell through TCR-MHC interaction ^ T cell interacts with B cell through TCR-MHC interaction and by CD40L-CD40 interaction-* IL-4 I I CD40L CD40 B cell activated and secretes IgM B cell acquires antigen B cell processes antigen and displays class II MHC-peptide complexes on surface Hypermutation Class switch recombination Other isotypes High affinity immunoglobulin 37 Antibodies as immunochemical tools The "tools": Immunology antibodies Immunoassay effector B cell (plasma cell) |_| 1 |xm 38 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. 0=C I I N-H lysine amino acid H -C -CHo-CH2-CH2-CH2-NH -^~"^-N02 m \ m dinitrophenyl | group (DNP) ■ H Hapten polypeptide other examP|es: backbone of toxins, pharmaceuticals, protein Carrier Definition of hapten: A low-molecular weight molecule which contains an antigenic determinant but which is not by itself antigenic unless bound to an antigenic carrier hormones. => Why do we need a carrier protein to launch an immune response against DNP? Cross reactivity (CR) Compound 6D8 8B1 10C9 ( 12G5 CR (in %) DCF 5-OH-DCF 4'-OH-DCF DCF-GLU Ibuprofen Ketoprofen Meclofenamic acid Fenoprofen Mefenamic acid Tolfenamic acid Cl OH OH Ck ^OH .OH Ck ^OH 100 100 100 100 3.5 9.6 10 13 6.2 1.7 5.3 11 why? 24 14 8.8 8.5 <0.42 <0.10 <0.43 < 0.0069 <0.42 <0.10 <0.43 < 0.069 <0.42 <0.10 <0.43 0.35 <0.25 <0.06 <0.43 < 0.25 2.4 0.74 <0.43 0.55 3.5 4.0 17 0.85 depends on antibody clone 0) O CD O CD CO I—i- c o CD CO o I—I-CD TD 0) CD CO Detection of diclofenac (DCF) by a competitive immunoassay 40 Polyclonal vs. monoclonal antibodies polyclonal monoclonal Antibodies that are collected Individual B cell hybridoma is cloned and from sera of exposed animal 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 generation: (1) Immunization of animal with pure antigen (immunogen) and with adjuvant (substance that strengthens the immune response) (2) Immunization is repeated (boost) (3) Collection of animal's blood (4) Purification of antibodies (IgG) Antibody production: fast and inexpensive 42 Choosing a host species for antibody production Common animals for obtaining antibodies: - Mice: easy breeding, but only small amounts - Rabbits: larger amounts => for polyclonal antibodies - Sheep/Goats: large amounts (commercial use) => for polyclonal antibodies => ease of breeding vs. antibody yields Other considerations for choosing a host species: - it is not possible to obtain anti-mouse IgG by immunizing mice (=> immunological tolerance) - if a mouse antigen is the target, mouse IgG may show cross-reactivity with other (non-target) mouse antigens 43 Polyclonal antibodies: -> and • Fast development, typically available first • Fast preparation • Inexpensive • Greater reagent versatility • 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 • Limited amounts (typically not sufficient for in excess reagent systems) • High batch-to-batch variability • Often lack full antigen specificity • Cannot discriminate between closely related antigens (potential for reduced specificity) • Pure antigen required for immunization 44 From polyclanal to monoclonal antibodies Until the 1970s, polyclonal antibodies were the only source of capture elements for immunochemical assays For the continuous supply of monoclonal antibodies (and their commercialization), we need plasma cells that live forever. Problem: B cells (like most body cells) can only undergo a limited number of cell divisions and die after a few days in cell culture 45 Monoclonal antibodies (Mab) Immunization Myelone Cell Culturt Why spleen (slezina) cells? first described in 1975 by Köhler/Milstein (Nobel prize 1984) HPRT- lq-Myelomo Cells (2xlOr) 'r n Selection of Hybrid Av ' Cell* in HAT Medium V-/ r* Assay for Antibody Clone Antibody-Producing (positive) Hybrids Tumor Induction Moss Culture Growth Freeze Hybridomo for Future Use Monoclonal Antibody Monoclonal Antibody 46 Generation of monoclonal antibodies mouse immunized with antigen X 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) resulting hybridoma celte cultured in multiple wells Generation of monoclonal antibodies only hybridoma cells survive and proliferate in the selective medium (HAT) secreted WW supernatant tested for anti-X antibody, and cells from positive well cultured at ~1 cell per well fTTTTTTTT Fl limiting dilution cells allowed to multiply, and individual supernatants tested for anti-X antibodies positive clones provide a continuing source of anti-X antibody Do you remember typical names of antibody clones? (E2/G2, B7, etc.) => Often simply indicate from which well the 48 clone was selceted Generation of monoclonal antibodies 1. Hyperimmunize mouse with antigen and adjuvant (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:4 Cell type: TK- immortal HAT-sensitive plasmacytoma TK+/TK - fused ^ hybrid TK + 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 49 *HGPRT (hypoxanthine-guanine phosphoribosyltransferase) mutants can be used in place of TK (thymidine kinase) mutants Expand in mice or in vitro 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 50 Monoclonal antibodies: -> and • Constant supply of the same antibody (from in vitro culture) • Consistent performance: constant affinity and specificity • 100 % epitope specificity __Jj • IgG fraction yields in practice an almost 100 % active antibody preparation • Enables the design of very specific assays for closely related antigens, and posttranslational variants (fragments, cleaved forms, sugar variants etc.) • Does not require 100 % antigen purity for immunization • Longer time for development • Expensive • Often of lower affinity than polyclonal antibodies => especially important if used in a competitive assay (in sandwich assay antibody excess compensates for lower affinity) • Can be too specific (do not recognize a genetic or other variant) 51 Monoclonal antibodies can be too specific if there is a common genetic variant of a protein wild-type ß I a ) protein "mutant" genetic variant of protein -> false negative result! 52 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! 53 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) 54 Labeling of antibodies with fluorescent dyes Structure R 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 56