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R. L. L. OSWE. P. ARK. L. aboratory. Cancer Institute. of. Flow. Cytometry. ISAC XX TUTORIAL Carleton C. Stewart Sigrid J. Stewart. RPCI. LFC. ANTIBODY BINDING TO CELLS. ANTIBODY STRUCTURE. Light Chain Kappa or Lambda. papain. pepsin. Fab. s s. Fc. s s. s s. Fab. s

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  1. R L L OSWE P ARK L aboratory Cancer Institute of Flow Cytometry ISAC XX TUTORIAL Carleton C. Stewart Sigrid J. Stewart

  2. RPCI LFC ANTIBODY BINDING TO CELLS

  3. ANTIBODY STRUCTURE Light Chain Kappa or Lambda papain pepsin Fab s s Fc s s s s Fab s s Heavy Chain IgG1 IgG2a IgG2b IgG2c IgG3 IgM IgA 1000 EPITOPES/ANTIGEN IgD 2 LIGHT CHAINS 10 HEAVY CHAINS IgE 20,000 CLONES

  4. Kf Ab + Ep AbEp Kr RATE f = Kf RATE r = Kr X[AbEp] X[Ab] [Ep] THE LAW OF MASS ACTION Kf range is usually ~ 106 Kr range is usually ~ 10-3 Ka = Kf/Kr = 106/10-3 = 109 AT EQUILIBRIUM RATE f = RATE r = 0 and [AE]=Ka[Ab][Ep]/(1+Ka[Ab])

  5. RPCI LFC WAYS ANTIBODIES BIND TO CELLS Specific: Fab to epitope Fc to Fc receptor binding is high affinity and saturable Non Specific: binding is low affinity and not saturable

  6. [F(ab') ] RPCI LFC Specific Activity is the concentration of bindable antibody to its epitope divided by the protein concentration. 2 SA = (protein)

  7. RPCI LFC Reasons Antibodies do not bind to cells: • overconjugation • not purified • degradation of binding site • aggregation

  8. RPCI LFC STORING OF ANTIBODIES : Proteases destroy antibodies in: • ascitic fluid • serum • bacteria Use sodium azide Use highly purified albumin or gelatin as carrier Purify antibodies immediately

  9. Ka=1011 Ka=1010 Ka=109 Ka=108 Ka=107 1 x 10-11 1 x 10-10 1 x 10-9 1 x 10-8 1 x 10-7

  10. 100 80 percent free 60 40 1010 107 1011 108 109 - - - - EFFECT OF AFFINITY CONSTANT ON ANTIBODY BINDING Affinity Constant (L/M)

  11. ANTIBODY BINDING TO MEMBRANE ANTIGENS 1000 MEASURED BINDING 10 1 SPECIFIC BINDING AMOUNT BOUND (ng/ml) Ka = 5 x 108 0.1 NON-SPECIFIC BINDING 0.01 Ka = 5 x 104 0.001 0.0001 0.01 0.1 1 10 100 1000 ANTIBODY (mg/ml)

  12. ANTIBODY BINDING TO INTRACELLULAR ANTIGENS MEASURED BINDING SPECIFIC BINDING AMOUNT BOUND (ng/ml) Ka = 5 x 108 NON-SPECIFIC BINDING Ka = 5 x 104 ANTIBODY (mg/ml)

  13. ANTIBODY BINDING TO INTRACELLULAR ANTIGENS HIGH AFFINITY ANTIBODY 10 2 /ml) MEASURED BINDING 10 ng 1 SPECIFIC BINDING 0.1 9 Ka = 5 x 10 AMOUNT BOUND ( 0.01 NON-SPECIFIC BINDING Ka = 5 x 10 4 0.001 0.0001 0.001 0.01 0.1 1 10 100 1000 m ANTIBODY ( g/ml)

  14. SPECIFIC AND NON-SPECIFIC ANTIBODY BINDING (MEMBRANE) Specific Binding high affinity Ka = 108 - 1010 for Ka = 5 x 108 saturation 1.0 µg/ml (0.1 µg/test) Non-Specific Binding low affinity Ka < 105 for Ka = 5 x 104 saturation 90 µg/ml (9 µg/test)

  15. SPECIFIC AND NON-SPECIFIC ANTIBODY BINDING (INTRACELLULAR) Specific Binding high affinity Ka = 108 - 1010 for Ka = 5 x 108 saturation 0.1 µg/ml (.01 µg/test) Non-Specific Binding low affinity Ka < 105 for Ka = 5 x 104 saturation 90 µg/ml (9 µg/test)

  16. TITERING ANTIBODIES RPCI LFC

  17. MEMBRANE EPITOPES RPCI LFC

  18. 3 µg s/n = 2.5 1 µg s/n = 2.1 0.3 µg s/n = 2.4 0.1 µg s/n = 4.1 0.03 µg s/n = 4.8 0.01 µg s/n = 4.6 3 0.003 µg s/n = 3.5 0.001 µg s/n = 3.2 auto

  19. 5 4 3 2 0 1 2 3 4 5 6 7 8 MEMBRANE ANTIGENS TITER Signal to Noise Dilution

  20. MEMBRANE ANTIGENS non-specific and specific binding titer ratio ng/test

  21. Verification of Specific Binding NUMBER OF CELLS CD4 FITC

  22. INTRACELLULAR EPITOPES RPCI LFC

  23. FACTORS THAT AFFECT DETECTION AFTER FIXATION • destruction of epitope by denaturation • loss of antigen by extraction • masking epitope by cross-linking • non-specific binding due to low affinity antibody by manufacture • cross-reacting products by GOD

  24. VERIFYING ANTIBODY SPECIFICITY CELLS • positive target cell lineage for antibody • genotypically identical negative target cell lineage • several different surrogate positive target cell lineages • several different negative target cell lineages

  25. VERIFYING ANTIBODY SPECIFICITY PROCESS • western blot • evaluation of Ka

  26. In the beginning there is a high extracellular antibody concentration.

  27. Antibodies diffuse relatively fast into the cell down the electrochemical gradient and bind specifically and non-specifically.

  28. BARRIERS TO INTRACELLULAR STAINING • immobilized epitope decreases antibody binding affinity • molecular crowding limits diffusion • fixation induced epitope degradation • fixation induced epitope obstruction

  29. BARRIERS TO SPECIFIC BINDING • molecular crowding increases non-specific binding • NSB reduces effective antibody concentration • washing efficiency reduced due to slow off-rate • multiple targets specificity with different epitope affinities (e.g. multiple bands on a western blot)

  30. So, what is the answer? Only one. Antibodies of high affinity so that there is a low free antibody concentration to begin with!

  31. 1 2 3 4 1 2 3 4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 1 1 2 2 3 3 4 4 INTRACELLULAR ANTIGENS isotype control antibody number cytokeratin 1 µg MCF Ab 278 IC 5.8 .3 µg MCF Ab 100 IC 3.6 .01 µg MCF Ab 25.7 IC 2.6

  32. 60 50 40 30 20 10 0 0 1 2 3 4 5 6 INTRACELLULAR ANTIGENS TITER Signal to Noise Dilution

  33. ANTIBODY BINDING RPCI LFC

  34. Differing Monoclonal Antibody Epitope Binding PE-CD19 SIDE SCATTER

  35. Importance of Fluorochrome Intensity FITC-CD69 PE-CD69 CD3 CD3

  36. Effect of the Drug Brefeldin A on Membrane CD69 Expression With BFA No BFA CD69 CD69 CD4 CD4

  37. TANDEM DYES RPCI LFC

  38. Variation In Compensation For PE-CY5 Reagents PE-fluorescence

  39. Effect Of Light Exposure on PECY5 Tandem Fluorescence 8 hours in dark PE-fluorescence 8 hours in light TC-CD3 TC-CD45

  40. A B C CD25 CD25 SSC PerCP-CD4 PECY5-CD4 FSC PECY5 Binding to Monocytes

  41. BLOCKING IS IMPORTANT RPCI LFC

  42. Primary Antibody: murine monoclonal antibody Fab Fab Fab Fab Fab Fc Fc Fc Fc Fc A B C D DIRECT IMMUNOFLUORESCENCE STAINING NO BLOCKING %+=75 FcR epitope

  43. Primary Antibody: murine monoclonal antibody Fab Fab Fab Fab Fab Fc Fc Fc Fc Fc A B C D DIRECT IMMUNOFLUORESCENCE STAINING NO BLOCKING %+=75 FcR epitope

  44. ISOTYPE CONTROL- myeloma protein %+=50 %+=75%-50=25% AUTOFLUORESCENCE CONTROL %+=0

  45. Fab Fab Fc Fc A B C D BLOCKING WITH GOAT IgG goat IgG FcR epitope %+=50

  46. F F F F F m m m m m m m m m m Primary Antibody: Second Antibody: murine monoclonal fluoresceinated antibody goat anti-mouse V V FcR epitope Fab Fab Fab Fab Fab Fc Fc Fc Fc Fc m m m m m INDIRECT IMMUNOFLUORESCENCE STAINING NO BLOCKING IgG F(ab')2

  47. F F F F F m m m m m m m m m m Primary Antibody: Second Antibody: murine monoclonal fluoresceinated antibody goat anti-mouse V V FcR m m m m m epitope Fab Fab Fab Fab Fab Fc Fc Fc Fc Fc m m m m m INDIRECT IMMUNOFLUORESCENCE STAINING NO BLOCKING IgG F(ab')2 directly labeled primary

  48. m m m m m F F F F F m m m m m m m m m m Primary Antibody: Second Antibody: murine monoclonal fluoresceinated antibody goat anti-mouse V V FcR epitope Fab Fab Fab Fab Fab Fc Fc Fc Fc Fc m m m m m INDIRECT IMMUNOFLUORESCENCE STAINING BLOCKING with mIgG IgG F(ab')2

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