Summary – directly labelled antibodies - No non-specific binding from secondary antibodies; - Fewer incubations and wash steps; - Better data quality; - Better reproducibility.

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Methods of antibody conjugation -
which one is best for you?

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Dr Nick Gee
CEO/CSO of Innova Biosciences
Inventor of Lightning-Link®

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Topics to be covered:
• Indirect and direct detection methods
• Reactive groups and cutting through the jargon
• Key methods for making antibody conjugates
• Key considerations before you start
• Innova conjugation technologies and applications

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Antigen
Primary Ab
Secondary Ab
Label
Primary Ab
Differences between direct and indirect detection

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Why should you directly label your antibody?
Mouse GoatRabbit
Anti-mouse Anti-rabbit

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Simplicity of direct detection
Mouse MouseRabbit

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• Fewer incubations and wash steps
• No non-specific binding from secondary antibodies
• Better data quality
• Better reproducibility
Summary – directly labelled antibodies

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Reactive group (B) on the label forms a covalent bond with a
reactive group (A) on the antibody.
LabelBA
Conjugation of antibody to another molecule (label)

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Labels with reactive ‘B’ groups are widely available; the key decision
is which B group to use in your reaction
LabelBA
Conjugation of antibody to another molecule (label)
Enzyme
Dye
Biotin
Streptavidin
Fluorescent protein
Particle
?

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Functional groups
in antibodies
Tyrosine
Lysine
Glutamate
Aspartate
Methionine
Serine
Histidine
Arginine
SAMPT
SIAB
DTNB
WRK
CDI
DST
DCC
SMCC
DFA
APDP
Conjugation chemicals
Maleimide
NHS (succinimidyl)
Iodoacetyl
Bromoacetyl
Sulfonyl chloride
Aldehyde
Chloromethyl
Alkyne
Thiol
Azide
Amine
EDC
DIC
CMC
SPDP
2-IT
SAMSA
SATA
ASIB
SAMCA
SAPB
SASD
PDPH
SIAC
DSG
NaIO4
MPBH
DMS
ADH
DTTA
SBED
LabelB
Most of these possibilities complicate what is actually very easy

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Tyrosine
Lysine
[Glutamate]
[Aspartate]
Methionine
Serine
Histidine
Arginine
SAMPT
SIAB
DTNB
WRK
CDI
DST
DCC
SMCC
DFA
APDP
Maleimide
NHS (succinimidyl)
Iodoacetyl
Bromoacetyl
Sulfonyl chloride
Aldehyde
Chloromethyl
Alkyne
Thiol
Azide
Amine
EDC
DIC
CMC
SPDP
2-IT
SAMSA
SATA
ASIB
SAMCA
SAPB
SASD
PDPH
SIAC
DSG
NaIO4
MPBH
DMS
ADH
DTTA
SBED
What you actually need to know ….

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Three key reactions….
2. Reaction of an amine with a carboxyl
1. Reaction of an NHS ester with an amine
3. How to thiolate an antibody
You can conjugate any antibody to any label
with just this knowledge

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For labelling antibodies with fluorescent dyes; simple, no
modification of antibody; Cons, NHS esters are very unstable
1. Reaction of an NHS ester with an amine (pH 8.0)
Lysine-NH2 Lysine-NH-C
+
R = Any dye
e.g. Fluorescein
+
NHS
O
R
N
O
O
R
O
O
N
O
OH
O

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For labelling antibodies with carboxylated particles; simple, no
modification of antibody; Cons, Ab-Ab conjugation
2. Reaction of an amine and carboxyl (pH 6.0)
Lysine-NH2 ParticleHOOC+
Carbodiimide
(EDC)
Lysine-NH-C
Particle
O

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To allow reaction of antibodies with maleimide-activated
protein labels; Cons; modification of antibody is required
i.e. separation steps to remove excess reactants
3. How to thiolate an antibody
lysine
+
S
NH
R N
SH
NH2
lysine
+
Lots of thiolation reagents – 2-IT is the easiest and best
2-iminothiolane

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Heterobifunctional reagents
Protein
Label
B
Used in the production of activated labels
B NHS ester
Protein
Label
NH2
e.g. SMCC
(adds maleimide)

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Y
Activation step
(lysine modification)
Y
Y
Y
Desalting or
Spin column
Antibody activation procedures
‘A’ group
NHS ester
e.g. for adding bromoacetyl groups, azides, alkynes,
hydrazino ligands, aromatic aldehydes.

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Why antibody activation steps are to be avoided..
Issue with all antibody modification procedures: losses of
antibody, variability, lack of scalability (up and down)
Spin column
Loss of materialCertainty Uncertainty
Activate

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LabelBAlysine
Need a ‘B’ group and technology that is compatible
with antibodies in their native unmodified form
XXXX
Choose functional groups that do not force you into
completely unnecessary modification of the antibody

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Label
Lightning-Link ®: a unique one-step conjugation technology
1. Activation (lys)
2. Conjugation
3. Quenching
4. Decay

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Lightning-Link® One-step
30 seconds hands-on time
No spin columns
No losses of antibody
Freely scalable
50 labels in kit format:
HRP, fluorescent
proteins, dyes,
Streptavidin…
All freeze dried
Westerns
ELISA
Flow Cytometry
Immunohistochemistry
And many more..

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Easier conjugations, better performance
Titration of conjugates
in ELISA
100 1000 10000 100000
0.00
0.25
0.50
0.75
1.00
1.25
1.50
Standard method
Lightning Link
Conjugate dilution factor
Absorbance(A405)

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2 3 4 5 6 7
0.0
0.5
1.0
1.5
2.0
2.5
50g
500g
5,000g
50,000g
Log10 Dilution Factor
Absorbance(A405)
Amount of antibody
Process simplicity - ease of scaling up/down

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Other key considerations – before you begin…
Check the buffer formulation of the antibody!
Glycine, histidine
Tissue culture media
Crude ascites fluid
Azide
BSA
Tris
!
Lysine reactions
+ EDTA (carboxyls)
(if carbodiimide is used)!
+ DTT, mercaptoethanol
!

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Label Reactive group Reacts
with
Ab modification
+ spin columns
Dye NHS Lysine NO
Particle COOH Lysine NO
Protein Maleimide Thiol YES (lysine)
All dyes &
proteins
Lightning-Link® Lysine NO
Summary of the easiest chemical approaches

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Avoid technologies that force you to modify your
antibody. You can couple labels directly to the lysines
that are already present on the antibody!
Avoid procedures with spin columns or other separation
steps – you will lose valuable antibody and introduce
variability.
Main messages:
Complex chemistries are not needed to make conjugates

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Contact
If you would like any more information, please contact us at
info@innovabiosciences.com
Please keep an eye out for our future webinars and other exciting news on our
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www.innovabiosciences.com/innova/webinars.html
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Innova Biosciences Ltd.
Babraham Research Campus,
Cambridge, UK,
CB22 3AT
www.innovabiosciences.com
Lightning-Link® is a registered trademark of Innova Biosciences
DyLight® is a registered trademark of Thermo Fisher Scientific Inc. and its subsidiaries