8 Applications and advantages of alkaline phosphatase in immunoassays

© Innova Biosciences ltd. 2013. All rights reserved
Quality – Consistency – Expertise

Welcome to our eighth webinar
Applications and advantages of alkaline
phosphatase in immunoassays
Dr Andy Lane

Dr Andy Lane
• Nature and characteristics of AP
• Advantages of AP in immunoassays
• Choice of substrates available
• How to prepare antibody:AP
conjugates

Alkaline Phosphatase -
nature and characteristics
AP is a 140kD enzyme, with a dimeric
structure.
Each sub-unit binds two Zn++ ions, one very
tightly, and a second, which is involved in
the catalytic reaction less tightly.
Mg++ ions are also bound, at different sites,
and stimulate catalysis. Excess Zn++ may
bind to the Mg++ sites and inhibit activity

Nature and characteristics
The alkaline phosphatase family is widely distributed, and functions to hydrolyze
phosphate groups from nucleotides and proteins.
In mammals there are two key forms. One is widely distributed, and the other is
restricted to the intestine. They react differently to inhibitors – tissue AP is inhibited
by 1mM levamisole, whilst intestinal AP is not.
The most commonly used AP in immunoassay and IHC applications is calf intestinal
AP. However, sources from other tissues such as placenta and recombinant
materials are also available.

Advantages in immunoassays and IHC
In contrast to enzymes such as HRP, AP reaction rates remain linear, and
therefore assay sensitivity can be increased by the very simple step of
increasing the reaction time with the substrate.
AP is also not inhibited by by-products of the substrate reaction, which can
be an issue when using HRP.
AP activity is not affected by exposure to the most commonly used
antibacterial agent, sodium azide, which strongly inhibits HRP.
In IHC, AP offers a very useful alternative to HRP where tissues have high
levels of endogenous peroxidase activity. Any endogenous AP activity can
simply be blocked by levamisole.

Choice of substrate
Chromogenic
Chromogenic substrates are available in two main forms – precipitating, as used
for IHC and WB, and soluble, as used for ELISA.
Chromogenic substrates are the simplest and most cost-effective substrates available, but
have lower sensitivity than other substrates and are therefore best suited to assays
where protein expression is high. An advantage of these substrates is that it is possible
to monitor colour development, and therefore it is easier to optimise conditions for
an assay.
Typical precipitating substrates for AP are BCIP and NBT, which provide a blue/purple
read-out. The most commonly used soluble substrate is PNPP, which provides
a yellow signal.

Choice of substrates
Chemiluminescent
Chemiluminescent substrates have the advantages of high sensitivity and a very wide
dynamic range. The light emitted by such substrates is only present when the
enzyme/substrate reaction is occurring, and no permanent coloured product is produced.
A key group of chemiluminescent substrates for AP are derivatives of 1,2-dioxetane.
Using these substrates sensitivity in the attomole range is achievable, and there are
reports of single molecule detection in 100ul samples using some derivatives
of these substrates.

Choice of substrates
Fluorescent
Fluorescent substrates are also available for use with AP, and also demonstrate high
sensitivity. Typical substrates include 4-MUP, and the more sensitive BBTP which
provides sensitivity in the attomole range.
An interesting substrate is that contained in the DuoLuX TM product, which has both
chemiluminescent and fluorescent properties. The fluorescence of this product may
be measured months after the luminescence has faded, providing a long term read-out
that may be especially useful for applications such as Western blot.

Conjugation of AP to antibodies
Whilst AP has a number of advantages over other enzymes that are commonly used
in immunoassays, it is a relatively difficult enzyme to work with.
It is not unusual to find losses of enzyme activity following conjugation. The specific
activity of AP can also vary widely from batch to batch, and several suppliers actually offer
AP at different grades. At a very basic level the fact that AP is colourless makes handling
more difficult.
Traditional conjugation techniques to link AP to antibodies or other proteins include
cross-linking with homofunctional linkers such as glutaraldehyde, and the application
of heterobifunctional cross-linkers such as SPDP and SMCC. Periodate based conjugation
has also been reported.

Conjugation of AP to antibodies
Glutaraldehyde based conjugation is an extremely difficult reaction to control, and leads to
great difficulty in preparing consistent conjugates.
In contrast methods using heterobifunctional linkers provide better control, but require
significant experience of chemistry and understanding of the variables that may affect
the reaction, as well as expensive protein purification systems to purify the
resulting conjugates.
A much simpler option is offered by the Lightning-Link® conjugation kits offered by
Innova Biosciences

What is Lightning-Link® technology?
The worlds fastest, easiest to use and most efficient conjugation technology!
• Only 30 seconds hands-on time!
• Over 50 labels available including:
Enzymes, fluorescent proteins / dyes, tandems, biotin & streptavidin
Lightning-Link®
Antibodies – Proteins – Peptides
Fast – Easy-to-use – Reliable

What is Lightning-Link® technology?
• 100% antibody recovery
• Fully scalable from R&D (10µg<) to Production / Manufacture (>1g)
• Virtually eliminates batch to batch variability
• Coupling of the label to antibodies, proteins or other biomolecules
• Covalent conjugation ensures long term stability
• Available as traditional Lightning-Link® (3 hour incubation) or new Lightning-Link®
RAPID (15 minute incubation)
Lightning-Link®

Alkaline Phosphatase anti-Human IgG
(Fc) Conjugates Analysed By ELISA
100 1000 10000 100000 1000000
0.0
0.5
1.0
1.5
2.0
2.5
3.0 LL Kit generated
secondary antibody
LL Kit generated
antibody with no antigen
Commercially available
secondary antibody
secondary antibody
with no antigen
Conjugate dilution
Absorbanceat405nm
Alkaline Phosphatase anti-Human IgG
(Fc) Conjugates Analysed By ELISA
(conjugate at 1:10000 dilution)
10 100 1000
0.0
0.5
1.0
1.5
2.0
LL Kit generated
secondary antibody
secondary antibody
Antigen / well (ng)
Absorbanceat405nm
Anti-human IgG monoclonal
antibody was purchased pre-
conjugated to alkaline phosphatase
from a commercial supplier. The
same antibody was purchased in
un-conjugated form and then
conjugated to alkaline phosphatase
using a Lightning-Link® kit.
The Lightning-Link® conjugate
demonstrates enhanced titre and
sensitivity.
Lightning-Link® Comparative Data

Conjugation considerations
You need to know some things about your reagent.
Lightning-Link conjugations are really simple
but you need protein in the right format to work effectively.
Concentration – 1mg/ml or higher is preferred
Purity – ensure other proteins have been removed,
and also make sure they haven’t been put back again afterwards!
Buffer formulation – most common formulations are suitable,
but ensure that amines such as glycine are truly absent,
as well as thiols such as DTT or mercaptoethanol. Tris is OK up to 20mM
Lightning Link kits are optimised for antibody labelling, but can easily be
adjusted to label other proteins. If you know the size of your protein you can
calculate how to use Lightning-Link for your application

Lightning-Link® increases the application of
AP in immunoassays
The excellent results demonstrated by Lightning-Link AP kits, combined with the
remarkable ease of use and reproducibility has led to a rapid and sustained increase
in the uptake of these kits by researchers and commercial assay manufacturers.
Alongside our catalogue offerings we are pleased to make available bespoke conjugation
kits to suit individual needs, and also to provide custom conjugation services.
“Lightning–Link® Alkaline Phosphatase kit is a simple, easy-to-use kit, to biochemically label protein
of interest, in our case, anti-HIV envelope antibodies, for ELISA. Labeling antibody with this kit
does not affect the function and avidity of the antibody. One advantage in using this kit is that
there is no need to remove excess alkaline phosphatase from the reaction mixture at the end
of the incubation. The omission of this step does not affect our ELISA analysis."
Tommy Tong, Post-Doctoral Fellow

Booth 3708 Booth 1E04

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
website and social media channels:
www.innovabiosciences.com/innova/webinars.html
YouTube: www.youtube.com/InnovaBiosciences

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

8 Applications and advantages of alkaline phosphatase in immunoassays

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