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Application of Antibodies in Purification and Analysis

Learn about antibodies, their synthesis, and applications in bioseparation and immunoassays. Discover how antibodies are synthesized in vivo and utilized in affinity chromatography and ELISA. Explore the genetic mechanisms diversifying antigen repertoires and antibody production via hybridoma cells. Delve into the immunoresponse, antibody therapy, and drug delivery. Gain insights into the specific detection and separation processes in bioprocesses like affinity chromatography. Uncover the principles of immunoassays, including RIA, EIA, and LIA, and the techniques and methods crucial in enzyme immunoassays.

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Application of Antibodies in Purification and Analysis

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  1. Application of antibodies in purification and analysis What is antibody ? How is antibody synthesized in vivo ? Immunoaffinity chromatography Enzyme linked immunosorbent assay (ELISA）

  2. (Variable region) Light chain (Constant region) Heavy chain What is antibody ? Antibody (IgG type) VH VL CH1 CL CH2 CH3 Protein secreted from lymphocytes (B cell, plasma cell) Several classes, IgG MW about 150000 Binding with many kinds antigens (proteins, sugars, toxins, virus, microorganisms and so on)

  3. (Variable region) Light chain (Constant region) Heavy chain Antibody (IgG type) VH VL CH1 CL CH2 CH3 Variable region Antigen binding site ------- Binding with wide variety of antigens Complementarity-determining region, CDR Constant region Opsonization, Compliment activation, Activation of NK cell Large number of repertoire and specificity against antigens

  4. Why antibodies can bind with wide variety of antigens ? Huge number of repertory Hypervariable regions gene variation CDR regions in VH and VL Rearrangement of gene in B and T cells What is the genetic mechanism of diversification of antigen repertoire ?

  5. antibodies Clonal selection of B cell repertoire by antigen

  6. Preparation of hybridoma cells producing a monoclonal antibody • Monoclonal antibody • Homogeneous antibody that recognizes a single type of antigenic site 1. B cells from an immunized animal are fused with immortal lymphocytes called myeloma cells. 2. In HAT medium, the unfused cells and the myeloma cells do not grow. The B cells have a limited life span in culture. Only fused cells formed from a myeloma cell and a B cell (hybridoma cells) can survive in HAT medium. 3. Each selected hybridoma cell is tested for the production of the desired antibody. (containing hypoxanthine, aminopterin, and thymidine)

  7. Amino acid variation in variable region

  8. Fab and lysozyme (yellow L-chain)

  9. How is antibody synthesized in vivo ? Immunosystem The body is protected from infectious agents and from other harmful substances. Humoral immunity Immunity mediated by antibodies Neutralization of toxin, marking of bacterial cells and virus Cell-mediated immunity T lymphocytes Killing infected cells Activate to effector cells

  10. Immunoresponse

  11. Application of antibodies Drug delivery, antibody therapy Neutralization of toxin, marking of bacterial cells and virus Targeting therapy Immunoassay Specific detection from complicated mixture

  12. Bioseparation (affinity chromatography) Specific adsorption washing Elution High purity separation in bioprocesses

  13. Affinity chromatography

  14. Secretion of a-amylase from recombinant yeast

  15. a-amylase SDS-PAGE of secreted of a-amylase in supernatant

  16. Separation process of secreted protein

  17. Chromatogram of affinity separation

  18. Purified a-amylase Crude broth

  19. Difference in molecules × binding non binding Assay of biological materials by use of antigen (Immunoassay) • Advantages of immunoassay • Specificity and high affinity • --- high sensitivity assay from mixture • Recognition subtle difference in similar molecules

  20. Immunoassay Radioimmunoassay (RIA) Enzyme immunoassay (EIA) homogeneous enzyme immunoassay heterogeneous enzyme immunoassay (Enzyme linked immunosorbent assay (ELISA)) Liposome immunoassay (LIA) Flow-injection analysis

  21. Classification of enzyme immunoassay Solid phasecompetitivetwo antibody solid phase method a) non-competitivedirect method b) sandwich method c) forward sandwich method simultaneous two site method liposome immunoassay Homogeneouscompetitive enzyme multiple immunoassay technique d) non-competitive liposome immune lysis assay

  22. Antigen in sample Enzyme labeled antigen E E E E Enzyme labeled antibody Adsorbed antibody Enzyme labeled hapten (a) (b) (d) (c) Schemes of enzyme immunoassay

  23. E Enzyme-conjugated antibody Relation between signal intensity and concentration

  24. Enzyme labeled antigen E Signal, arbitrary unit Antigen in sample Relation between signal and sample concentration in competitive methods Signal from enzyme labeled antigen Amount of sample antigen coupled to antibody

  25. * Enzyme immunoassay Enzyme linked immunosorbent assay (ELISA) Liposome immunosorbent assay (LISA) Color signal depended on antigen concentration

  26. Enzyme Immunoassay Enzyme conjugated antibody against target antigen Enzyme-substrate reaction Washing Substrate

  27. Antigen Blocking of surface Blocking agent Adsorption of primary antibody Primary antibody Adsorption of labeled antibody Enzyme-conjugate antibody Color development Adsorption of antigen ELISA

  28. Washing Washing Immobilization of ligand protein Binding analyte Blocking Solid-liquid reaction B/F separation Washing Washing Long time consuming Multiple steps for detection Enzyme-conjugated antibody Detection Operation in sandwich ELISA

  29. Liposome Immunoassay Enzyme Immunoassay Enzyme-conjugated antibody Immunoliposome Few enzyme molecules conjugated with one molecule of antibody Large number of enzyme molecules encapsulated in a liposome

  30. Diameter of liposome ▲350 nm ▲200 nm ▲130 nm ▲60 nm ▲ELISA Coating antigen, human IgM concn, 10 mg/ml Immunoliposome, anti-rabbit IgG (goat) concn, 3mg/ml Merits of immunoliposome High signal intensity Low signal High signal

  31. * Liposome immunosorbent assay (LISA) Advantages of LISA high sensitivity high level of signal * Membrane blotting assay *automated micro-assay method labeled antibody (conventional) *limited sensitivity and strength of signal Blotting assay using immunoliposomes

  32. HRP Immunoliposome Reaction products precipitated Substrate (4-chloro-1-naphthol) Primary antibody Antigen Membrane HRP-conjugated antibody Membrane conventional immunoblotting assay liposome immunoblotting assay color development (H2O2)

  33. Micro-membrane blotting Antigen blotting and blocking Immunoliposome reaction Primary antibody reaction Color development

  34. IgM concn, mg/ml 2.50 0.156 0.00977 0 Sample volume (ml) 10.0 0.625 0.0391 0.00244 10.0 2.50 0.313 0 20 20 20.0 5.00 1.25 0.0783 10 10 5 5 2 2 IgM concn, mg/ml Sample volume (ml) Comparison between liposome and conventional immunoblotting assays Immunoliposome HRP conjugated antibody

  35. Comparison between liposome and conventional immunoblotting assays Liposome Immunoblotting Assay Conventional Immunoblotting Assay

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