1. Técnicas de Imunologia Prof.Doutor José Cabeda Técnicas de Imunologia Citometria de Fluxo Slides adaptados de uma colecção amavelmente cedida por Alexandre Salvador (BioAtlântico)

2. Técnicas de Imunologia Prof.Doutor José Cabeda Marcação celular com Anticorpos em Citometria de Fluxo

3. Técnicas de Imunologia Prof. Doutor José Cabeda K f range is usually ~ 10 6 K r range is usually ~ 10 -3 K a = K f /K r = 10 6 /10 -3 = 10 9 KfKf KrKr Ab + Ep AbEp THE LAW OF MASS ACTION X[Ab] [Ep] RATE f = K f X[AbEp] RATE r = K r AT EQUILIBRIUM RATE f = RATE r = 0 and [AE]=Ka[Ab][Ep]/(1+Ka[Ab])

4. Técnicas de Imunologia Prof. Doutor José Cabeda Specific: Fab to epitope Fc to Fc receptor binding is high affinity and saturable Non Specific: binding is low affinity and not saturable WAYS ANTIBODIES BIND TO CELLS

5. Técnicas de Imunologia Prof. Doutor José Cabeda Specific Activity is the concentration of bindable antibody to its epitope divided by the protein concentration. 2 [F(ab')] SA = (protein)

6. Técnicas de Imunologia Prof. Doutor José Cabeda Reasons Antibodies do not bind to cells: overconjugation not purified degradation of binding site aggregation

7. Técnicas de Imunologia Prof. Doutor José Cabeda 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

8. Técnicas de Imunologia Prof. Doutor José Cabeda 1 x 10 -11 1 x 10 -10 1 x 10 -9 1 x 10 -8 1 x 10 -7 Ka=10 7 Ka=10 8 Ka=10 9 Ka=10 10 Ka=10 11

9. Técnicas de Imunologia Prof. Doutor José Cabeda EFFECT OF AFFINITY CONSTANT ON ANTIBODY BINDING Affinity Constant (L/M) 40 60 80 100 ---- 10 7 10 8 10 9 10 10 11 percent free

10. Técnicas de Imunologia Prof. Doutor José Cabeda 1000100 0.0001 0.001 ANTIBODY BINDING TO INTRACELLULAR ANTIGENS HIGH AFFINITY ANTIBODY ANTIBODY ( g/ml) AMOUNT BOUND ( ng /ml) 10 2 1 0.1 0.01 0.0010.010.1110 MEASURED BINDING SPECIFIC BINDING NON-SPECIFIC BINDING Ka = 5 x 10 4 Ka = 5 x 10 9

11. Técnicas de Imunologia Prof.Doutor José Cabeda TITERING ANTIBODIES

12. Técnicas de Imunologia Prof.Doutor José Cabeda MEMBRANE EPITOPES

13. Técnicas de Imunologia Prof. Doutor José Cabeda

14. Técnicas de Imunologia Prof. Doutor José Cabeda

15. Técnicas de Imunologia Prof. Doutor José Cabeda 876543210 2 3 4 5 Dilution Signal to Noise TITER MEMBRANE ANTIGENS

16. Técnicas de Imunologia Prof. Doutor José Cabeda MEMBRANE ANTIGENS ng/test ratioratio titer non-specific and specific binding

17. Técnicas de Imunologia Prof. Doutor José Cabeda CD4 FITC NUMBER OF CELLS Verification of Specific Binding

18. Técnicas de Imunologia Prof.Doutor José Cabeda INTRACELLULAR EPITOPES

19. Técnicas de Imunologia Prof. Doutor José Cabeda In the beginning there is a high extracellular antibody concentration.

20. Técnicas de Imunologia Prof. Doutor José Cabeda

21. Técnicas de Imunologia Prof. Doutor José Cabeda Antibodies diffuse relatively fast into the cell down the electrochemical gradient and bind specifically and non- specifically.

22. Técnicas de Imunologia Prof. Doutor José Cabeda

23. Técnicas de Imunologia Prof. Doutor José Cabeda BARRIERS TO INTRACELLULAR STAINING immobilized epitope decreases antibody binding affinity molecular crowding limits diffusion fixation induced epitope degradation fixation induced epitope obstruction

24. Técnicas de Imunologia Prof. Doutor José Cabeda 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)

25. Técnicas de Imunologia Prof. Doutor José Cabeda So, what is the answer? Only one. Antibodies of high affinity so that there is a low free antibody concentration to begin with!

26. Técnicas de Imunologia Prof. Doutor José Cabeda 10 1 2 3 4 1 µg MCF Ab 278 IC 5.8 isotype control antibody cytokeratin.3 µg MCF Ab 100 IC 3.6 10 1 2 3 4.01 µg MCF Ab 25.7 IC 2.6 number INTRACELLULAR ANTIGENS

27. Técnicas de Imunologia Prof. Doutor José Cabeda BLOCKING IS IMPORTANT

28. Técnicas de Imunologia Prof. Doutor José Cabeda DIRECT IMMUNOFLUORESCENCE STAINING NO BLOCKING Primary Antibody: murine monoclonal antibody A epitope FcR Fab Fc Fab Fc Fab Fc Fab Fc Fab Fc BCD %+=75

29. Técnicas de Imunologia Prof. Doutor José Cabeda ISOTYPE CONTROL- myeloma protein AUTOFLUORESCENCE CONTROL %+=50 %+=0 %+=75%-50=25%

30. Técnicas de Imunologia Prof. Doutor José Cabeda BLOCKING WITH GOAT IgG goat IgG A epitope FcR BCD Fab Fc Fab Fc %+=50