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

ANTIBODY BINDING TO CELLS RPCI LFC

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

K f range is usually ~ 10 6 K r range is usually ~ 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])

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 RPCI LFC

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

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

1 x x x x x Ka=10 7 Ka=10 8 Ka=10 9 Ka=10 10 Ka=10 11

EFFECT OF AFFINITY CONSTANT ON ANTIBODY BINDING Affinity Constant (L/M) percent free

ANTIBODY (  g/ml) MEASURED BINDING SPECIFIC BINDING NON-SPECIFIC BINDING Ka = 5 x 10 4 Ka = 5 x 10 8 ANTIBODY BINDING TO MEMBRANE ANTIGENS AMOUNT BOUND (ng/ml)

MEASURED BINDING SPECIFIC BINDING NON-SPECIFIC BINDING Ka = 5 x 10 4 Ka = 5 x 10 8 ANTIBODY BINDING TO INTRACELLULAR ANTIGENS ANTIBODY (  g/ml)

ANTIBODY BINDING TO INTRACELLULAR ANTIGENS HIGH AFFINITY ANTIBODY ANTIBODY (  g/ml) AMOUNT BOUND ( ng /ml) MEASURED BINDING SPECIFIC BINDING NON-SPECIFIC BINDING Ka = 5 x 10 4 Ka = 5 x 10 9

SPECIFIC AND NON-SPECIFIC ANTIBODY BINDING (MEMBRANE) Specific Binding high affinity Ka = for Ka = 5 x 10 8 saturation 1.0 µg/ml (0.1 µg/test) Non-Specific Binding low affinity Ka < 10 5 for Ka = 5 x 10 4 saturation 90 µg/ml (9 µg/test)

SPECIFIC AND NON-SPECIFIC ANTIBODY BINDING (INTRACELLULAR) Specific Binding high affinity Ka = for Ka = 5 x 10 8 saturation 0.1 µg/ml (.01 µg/test) Non-Specific Binding low affinity Ka < 10 5 for Ka = 5 x 10 4 saturation 90 µg/ml (9 µg/test)

TITERING ANTIBODIES RPCI LFC

MEMBRANE EPITOPES RPCI LFC

Dilution Signal to Noise TITER MEMBRANE ANTIGENS

ng/test ratioratio titer non-specific and specific binding

CD4 FITC NUMBER OF CELLS Verification of Specific Binding

INTRACELLULAR EPITOPES RPCI LFC

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 FACTORS THAT AFFECT DETECTION AFTER FIXATION

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 VERIFYING ANTIBODY SPECIFICITY CELLS

western blot evaluation of Ka VERIFYING ANTIBODY SPECIFICITY PROCESS

In the beginning there is a high extracellular antibody concentration.

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

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

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)

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

µg MCF Ab 278 IC 5.8 isotype control antibody cytokeratin.3 µg MCF Ab 100 IC µg MCF Ab 25.7 IC 2.6 number INTRACELLULAR ANTIGENS

Dilution Signal to Noise TITER INTRACELLULAR ANTIGENS

ANTIBODY BINDING RPCI LFC

SIDE SCATTER PE-CD19PE-CD19 Differing Monoclonal Antibody Epitope Binding

PE-CD69PE-CD69 FITC-CD69FITC-CD69 CD3 Importance of Fluorochrome Intensity

No BFA With BFA CD4 Effect of the Drug Brefeldin A on Membrane CD69 Expression CD69CD69 CD69CD69

TANDEM DYES RPCI LFC

PE-fluorescence Variation In Compensation For PE-CY5 Reagents

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

ABC PerCP-CD4 PECY5-CD4FSC SSCSSC CD25CD25 CD25CD25 PECY5 Binding to Monocytes

BLOCKING IS IMPORTANT RPCI LFC

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

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

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

BLOCKING WITH GOAT IgG goat IgG A epitope FcR BCD Fab Fc Fab Fc %+=50

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

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

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

add Mab Fab add fluoresceinated goat anti-mouse IgG F(ab') 2

VERIFICATION OF BLOCK 1. FcR and non-specific binding FL-MAB + PE-mIgG 2. goat IgG + FL-MAB + PE-mIgG

EFFECT OF BLOCKING ON ANTIBODY BINDING TO MONONUCLEAR CELLS LOG FLUORESCENCE CELL VOLUME

number TOTAL A B C D channel number

variation in gamma 1 myeloma protein binding to macrophages mopc myeloma protein

VIABILITY IS IMPORTANT RPCI LFC

ANTIBODIES BIND NON- SPECIFICALLY TO DEAD CELLS FL-KAPPA PE-LAMBDA dead cells ALL CELLSVIABLE CELLS AB

lysed, washed cells + 5 µg EMA 10 min. 18 cm. EMA PROCEDURE WASH, FIX, AND ANALYZE 1 3 2

EVALUATING VIABILITY WITH ETHIDIUM MONOAZIDE SSCSSC FSC % dead in gate = 2% FSC SSCSSC EMAEMA % dead = 12%

Ab1 Ab3Ab3 Ab2Ab2 AB Effect of Dead Cells and Antibody Affinity on Immunophenotyping

COMPENSATION RPCI LFC

emission intensity wavelength FLBFLA

-/\/\/\/\/\ FLBFLB A%B B%A 0 %B FLAFLA Opamp A optical filters PMT & preamp 20 %A Opamp B FLBFLB A%B B%A 5 %B FLAFLA Opamp A optical filters PMT & preamp 0 %A Opamp B -/\/\/\/\/\-

+ = FLBFLB FLAFLA A%B B%A 5 %B Opamp A 20 %A Opamp B + = + = + =

Uncompensated vs Compensated FL1 FL2

FL1 FL2 COMPENSATION IS INTENSITY DEPENDENT uncompensated partially compensated fully compensated FL2

COMPENSATE INSTRUMENT USING STAINED CELLS 1. Adjust PMT voltages using unstained cells 2. Adjust compensation for each fluorochrome

R1 R2 R3 R4 R5 R6

R3 A C B D

FSC SSC R1 R2R3 R4 FITC-CD45 PE-CD4 PE-CY5-CD8 PE-CD4 R4R5 R6 PE-CY5-CD8 APC-CD8 R8 R9 R10 COMPENSATION UP TO FOUR COLORS WITH TWO LASERS