R LLOSWEARKP

L Cancer Instituteaboratory

Flowof

Cytometry

ISAC XXISAC XX

TUTORIALTUTORIAL

Carleton C. StewartCarleton C. Stewart

Sigrid J. StewartSigrid J. Stewart

ANTIBODY BINDING TO CELLSANTIBODY BINDING TO CELLS

RPCI

LFC

FcFab

Fab

Light ChainKappa or Lambda

IgG1IgG2aIgG2bIgG2cIgG3

IgM

IgA

IgD

IgE

ANTIBODY STRUCTURE

1000 EPITOPES/ANTIGEN

2 LIGHT CHAINS10 HEAVY CHAINS

20,000 CLONES

Heavy Chain

ss

ss

ss

papain pepsin

ss

Kf range is usually ~ 106

Kr range is usually ~ 10-3

Ka = Kf/Kr = 106/10-3 = 109

Kf

Kr

Ab + Ep AbEp

THE LAW OF MASS ACTIONTHE LAW OF MASS ACTION

X[Ab] [Ep]RATE f = Kf X[AbEp]RATE r = Kr

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 CELLSWAYS 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 10-11 1 x 10-10 1 x 10-9 1 x 10-8 1 x 10-7

Ka=10Ka=1077

Ka=10Ka=1088

Ka=10Ka=1099

Ka=10Ka=101010

Ka=10Ka=101111

EFFECT OF AFFINITY CONSTANT ON ANTIBODY BINDING

Affinity Constant (L/M)

40

60

80

100

- - - -107 108 109 1010 1011

per

cen

t fr

ee

0.01 0.1 1 10 100 1000

ANTIBODY (g/ml)

1000

10

1

0.1

0.01

0.001

0.0001

MEASURED BINDING

SPECIFIC BINDING

NON-SPECIFIC BINDINGKa = 5 x 104

Ka = 5 x 108

ANTIBODY BINDING TO MEMBRANE ANTIGENSA

MO

UN

T B

OU

ND

(n

g/m

l)

0.001

0.01

0.1

1

10

100

1000

0.001 0.01 0.1 1 10 100 1000

AM

OU

NT

BO

UN

D (

ng/

ml)

MEASURED BINDING

SPECIFIC BINDING

NON-SPECIFIC BINDINGKa = 5 x 104

Ka = 5 x 108

ANTIBODY BINDING TO INTRACELLULAR ANTIGENS

ANTIBODY (g/ml)

1000100

0.0001

0.001

ANTIBODY BINDING TO INTRACELLULAR ANTIGENS

HIGH AFFINITY ANTIBODY

ANTIBODY ( g/ml)

AM

OU

NT

BO

UN

D (

ng

/ml)

10 2

10

1

0.1

0.01

0.001 0.01 0.1 1 10

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 = 108 - 1010

for Ka = 5 x 108 saturation1.0 µg/ml (0.1 µg/test)

Non-Specific Binding

low affinity

Ka < 105

for Ka = 5 x 104 saturation90 µg/ml (9 µg/test)

SPECIFIC AND NON-SPECIFIC ANTIBODY BINDING (INTRACELLULAR)

Specific Binding

high affinity

Ka = 108 - 1010

for Ka = 5 x 108 saturation0.1 µg/ml (.01 µg/test)

Non-Specific Binding

low affinity

Ka < 105

for Ka = 5 x 104 saturation90 µg/ml (9 µg/test)

TITERING ANTIBODIESRPCI

LFC

MEMBRANE EPITOPESRPCI

LFC

3

3 µgs/n = 2.5

1 µgs/n = 2.1

0.3 µgs/n = 2.4

0.1 µgs/n = 4.1

0.03 µgs/n = 4.8

0.01 µgs/n = 4.6

0.003 µgs/n = 3.5

0.001 µgs/n = 3.2

auto

8765432102

3

4

5

Dilution

Sig

nal

to

Noi

seTITER

MEMBRANE ANTIGENS

MEMBRANE ANTIGENS

1

1.2

1.4

1.6

1.8

2

2.2

300010003331113712410.50.20.0510.017

ng/test

ratio

titer

non-specific and specific binding

CD4 FITC

NU

MB

ER

OF

CE

LL

S Verification of Specific Binding

INTRACELLULAR EPITOPESRPCI

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!

101 102 103 104101 102 103 104101 102 103 104

1 µg MCFAb 278IC 5.8

isotypecontrol

antibody

cytokeratin.3 µg MCFAb 100IC 3.6

101 102 103 104

.01 µg MCFAb 25.7IC 2.6

nu

mb

erINTRACELLULAR ANTIGENSINTRACELLULAR ANTIGENS

65432100

10

20

30

40

50

60

Dilution

Sig

nal

to

Noi

seTITER

INTRACELLULAR ANTIGENS

ANTIBODY BINDINGRPCI

LFC

10 20 30 40 50 60 70 80 90 100 110 120

Side Scatter -->

10

11

02

10

31

04

Anti-T

CR

-alp

ha-b

eta

-1 F

ITC

-->

SIDE SCATTER

10 20 30 40 50 60 70 80 90 100 110 120

Side Scatter -->

10

11

02

10

31

04

Anti-T

CR

-alp

ha-b

eta

-1 F

ITC

-->P

E-CD19

Differing Monoclonal Antibody Epitope Binding

10 1 10 2 10 3 10 4

CD8 TC -->

101

102

103

104

Ant

i-TC

R-g

amm

a-de

lta-1

PE

-->

10 1 10 2 10 3 10 4

CD8 TC -->10

110

210

310

4

Ant

i-TC

R-g

amm

a-de

lta-1

PE

-->

PE-CD69

FITC-CD69

CD3 CD3

Importance of Fluorochrome Intensity

10 1 10 2 10 3 10 4

CD8 TC -->

101

102

103

104

Ant

i-TC

R-g

amm

a-de

lta-1

PE

-->

No BFA

10 1 10 2 10 3 10 4

CD8 TC -->10

110

210

310

4

Ant

i-TC

R-g

amm

a-de

lta-1

PE

-->

With BFA

CD4 CD4

Effect of the Drug Brefeldin A on Membrane CD69 Expression

CD69

CD69

TANDEM DYES

RPCI

LFC

PE

-flu

ores

cenc

eVariation In Compensation For PE-CY5 Reagents

PE

-flu

ores

cen

ce8 hours in dark

8 hours in light

TC-CD45 TC-CD3

Effect Of Light Exposure on PECY5 Tandem Fluorescence

A B C

PerCP-CD4 PECY5-CD4 FSC

SSC

CD25

CD25

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

B C D

%+=75%+=75

DIRECT IMMUNOFLUORESCENCE STAINING

NO BLOCKING

Primary Antibody:murine monoclonal antibody

A

epitope

FcR

Fab

Fc

Fab

Fc

Fab

Fc

Fab

Fc

Fab

Fc

B C D

%+=75%+=75

ISOTYPE CONTROL- myeloma protein

AUTOFLUORESCENCE CONTROL

%+=50%+=50

%+=0%+=0

%+=75%-50=25%%+=75%-50=25%

BLOCKING WITH GOAT IgG

goat IgG

A

epitope

FcR

B C D

Fab

Fc

Fab

Fc

%+=50%+=50

INDIRECT IMMUNOFLUORESCENCE STAINING

NO BLOCKING

Primary Antibody:murine monoclonal antibody

Second Antibody:fluoresceinatedgoat anti-mouse IgG F(ab')2

V

m

mF

VFcR

epitope

Fab

Fc

mm

mF Fab

Fc

m

Fab

Fc

m

m

mF

Fab

Fc

m

m

mF

Fab

Fcm

m

mF

INDIRECT IMMUNOFLUORESCENCE STAINING

NO BLOCKING

Primary Antibody:murine monoclonal antibody

Second Antibody:fluoresceinatedgoat anti-mouse IgG F(ab')2

V

m

mF

VFcR

epitope

Fab

Fc

m

mm

m

m m

mF Fab

Fc

m

Fab

Fc

m

m

mF

Fab

Fc

m

m

mF

Fab

Fcm

m

mF

m

directly labeled primarydirectly labeled primary

INDIRECT IMMUNOFLUORESCENCE STAINING

BLOCKING with mIgG

Primary Antibody:murine monoclonal antibody

Second Antibody:fluoresceinatedgoat anti-mouse IgG F(ab')2

V

m

mF

Fab

Fc

m

VFcR

epitope

Fab

Fc

mm

mF

Fab

Fcm

Fab

Fc

m

Fab

Fc

m

m

mF

m

mF

m

mF

m

m m

m m

add MabFab

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 CELLSL

OG

FL

UO

RE

SC

EN

CE

CELL VOLUME

nu

mb

erTOTAL

A

B

C

D

channel number

variation in gamma 1 myeloma protein binding to macrophages

05

101520253035404550

0 17 39 4 23 13 21

mopc myeloma protein

VIABILITY IS IMPORTANT

RPCI

LFC

ANTIBODIES BIND NON-SPECIFICALLY TO DEAD CELLS

FL-KAPPA

PE

-LA

MB

DA

dead cells

ALL CELLS VIABLE CELLS

A B

lysed, washedcells

+ 5 µg EMA

10 min.

18 cm.

EMA PROCEDURE

WASH, FIX, AND ANALYZE

1

3

2

EVALUATING VIABILITY WITH ETHIDIUM MONOAZIDE

10 20 30 40 50 60 70 80 90 100 110 120

Forward Scatter -->

010

2030

4050

6070

8090

100

110

120

Sid

e S

catte

r --

>

SSC

FSC

% dead in gate = 2%

10 20 30 40 50 60 70 80 90 100 110 120

Forward Scatter -->

01

02

03

04

05

06

07

08

09

01

00

110

12

0

Sid

e S

catt

er

-->

FSC

SSC

10 20 30 40 50 60 70 80 90 100 110 120

Side Scatter -->

10

11

02

10

31

04

Ant

i-TC

R-a

lpha

-bet

a-1

FIT

C -

->

FSC

EMA

% dead = 12%

Ab1

Ab3

Ab1

Ab2

A B

Effect of Dead Cells and Antibody Affinity on Immunophenotyping

COMPENSATION

RPCI

LFC

emission intensity

wavelength

FLBFLA

-/\/

\/\/

\/\

--/

\/\/

\/\

/\-

-/\/

\/\/

\/\

-

-

-

+

+FLB

A %B

B %A

0 %B

FLA

Opamp A

optical filters PMT & preamp

20 %A

Opamp B

-

-

+

+FLB

A %B

B %A

5 %B

FLA

Opamp A

optical filtersPMT & preamp

0 %A

Opamp B

-/\/

\/\/

\/\

-

+ =-/\/

\/\/

\/\-

-/\/

\/\/

\/\-

FLB

FLA

-

-

+

+

A %B

B %A

5 %B

Opamp A

20 %A

Opamp B

+ =

+ =

+ =

Uncompensated vs Compensated

FL1

FL

2

FL1

FL

2COMPENSATION IS INTENSITY

DEPENDENT

uncompensatedpartially

compensated

fully compensated

FL

2

COMPENSATE INSTRUMENT USING STAINED CELLS

1. Adjust PMT voltages using unstained cells

2. Adjust compensation for each fluorochrome

10 10 10 10

FITC-CD3

PE-C

D8

10

TC

-CD

2

FITC-CD3

PE-C

D8

TC-CD22 10 10

10 2 10 4

EC

D-C

D4

FITC-CD310 10 10 10

EC

D-C

D4

TC-CD2

10 2 10 10

PE-C

D8

ECD-CD4

UNCOMPENSATED

SOFTWARE COMPENSATION

101 102 103 104

10

1

10

2

10

3

10

4

PE

CD

8 --

>

PE

-CD

8

TC-CD2101 102 103 104

10

1

10

2

10

3

10

4

PE

-CD

8

ECD-CD4

101 102 103 104

10

1

10

2

10

3

10

4

TC

-CD

2

FITC-CD3101 102 103 104

10

1

10

2

10

3

10

4

EC

D C

D4

-->

EC

D-C

D4

FITC-CD3101 102 103 104

10

1

10

2

10

3

10

4

EC

D-C

D4

TC-CD2

101 102 103 104

10

1

10

2

10

3

10

4

PE

-CD

8

FITC-CD3

R1

R2

R3

R4

R5

R6

101 102 103 104

FITC CD3 -->

10

1

10

2

10

3

10

4

PE

CD

8 --

>

PE

-CD

8

FITC-CD3101 102 103 104

10

1

10

2

10

3

10

4

PE

CD

8 --

>

PE

-CD

8

TC-CD2

INSTRUMENT COMPENSATION

101 102 103 104

ECD CD4 -->

10

1

10

2

10

3

10

4

PE

CD

8 --

>

PE

-CD

8

ECD-CD4

101 102 103 104

10

1

10

2

10

3

10

4

TC C

D2

-->

TC-C

D2

FITC-CD3101 102 103 104

10

1

10

2

10

3

10

4

EC

D C

D4

-->

EC

D-C

D4

FITC-CD3101 102 103 104

10

1

10

2

10

3

10

4

EC

D-C

D4

TC-CD2

PE, TX-RED & PE-CY5COMPENSATION

TC CD2 -->101 102 103 10

10

1

10

2

10

3

10

PE

-CD

8

TC-CD2101 102 103 104

ECD CD4 -->

10

1

10

2

10

3

10

4

PE

-CD

8

ECD-CD4

POOR

GOOD

101 102 103 104

10

1

10

2

10

3

10

4

PE

-CD

8

TC-CD2101 102 103 104

ECD CD4 -->

10

1

10

2

10

3

10

4

PE

-CD

8

ECD-CD4

R3

R3

A C

B D

10 20 30 40 50 60 70 80 90 100 120

FSC -->

1020

3040

5060

7080

90100

120

SS

C -->

10 1 10 2 10 3 10 4

FL1 -->

101

102

103

104

FL2 --> R1

1 2

3 4

FSC

SSC

R1

R2 R3

R4

FITC-CD45

PE

-CD

4

10 1 10 2 10 3 10 4

FL4 -->

101

102

103

104

FL

2 --> R9

9 10

11 12

PE-CY5-CD8P

E-C

D4

R4 R5

R6

10 1 10 2 10 3 10 4

FL1 -->

101

102

103

104

FL3 --> R13

13 14

15 16

PE-CY5-CD8

AP

C-C

D8

R8

R9

R10

COMPENSATION UP TO FOUR COLORS WITH TWO LASERS