A Guide to Immuno-PCR

www.innovabiosciences.com
A Guide to Immuno-PCR

Our speaker today…
Emma Easthope
Technical Content Writer at Innova Biosciences

Agenda
• ELISA and real time PCR - advantages, disadvantages
• Main steps of Immuno-PCR assay
• Introduction to Thunder-Link® PLUS oligo labeling technology
• Q&A session

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Q&A session

What is immunodetection?
• Immunodetection is the process of using antibodies to detect
antigens in a sample
• Western blotting
• Immunocytochemistry
• Immunohistochemistry
• Flow cytometry
• ELISA
• Sensitive
• Selective
• Wide variety of analytes

What is immuno-PCR?
• Developed in 1992 by Sano et al
“Immuno-PCR: very sensitive antigen detection by means of specific antibody-DNA
conjugates” Science 258 (5079) 120-122
• Combines the antibody-based specificity of an ELISA with the
amplification of PCR
• Extremely high levels of sensitivity

ELISA
• Enzyme-Linked Immunosorbent Assay
• Evolved in the 1970s from radioimmunoassay (RIA)
• Relies on an antibody which has been linked to an enzyme
• Horseradish peroxidase, Alkaline Phosphatase, Glucose Oxidase
• Colorimetric readout
• Alternative readouts have been developed
• Fluorescence
• Luminescence

ELISA – advantages & disadvantages
Advantages Disadvantages
• Easy to perform
• Quantitative immunoassay
• Sensitive – limit of detection typically µg – pg
• Relatively inexpensive
• Can be used to screen large numbers of
samples
• Amenable to automation – suitable for High
Throughput Screening
• Cannot be used to measure parameters
such as tissue distribution or molecular
weight of the target
• The use of labeled secondary antibodies
for detection can introduce background
signal
• Some readouts are not amenable to
multiplexing

ELISA assay formats
• An ELISA can be performed via several methods

Lightning-Link® for direct antibody labeling
✓ Quick and easy to use
✓ Only 30 seconds hands-on time
✓ No separation steps - 100% recovery
✓ Scalable
Lightning-Link® labeling kits
• Enzymes – HRP, AP, Glucose oxidase
• Fluorescent dyes
• Fluorescent proteins
• Biotin
• Streptavidin
New product! Europium conjugation kit for covalent attachment of antibodies or proteins to
specially-treated 200nm europium particles – ideal for time resolved fluorescence (TRF)

Polymerase Chain Reaction (PCR)
• Invented in 1983
• Cyclical amplification of specific segments of DNA

PCR detection methods – gel electrophoresis
• Ethidium bromide was traditionally used to
visualize DNA following gel electrophoresis
• DNA interchelator
• Requires a UV transilluminator for visualization
• Molecular weight markers and restriction enzymes
can be used to confirm amplification of the target
• Southern blotting can provide additional
confirmation
DNA stained with EtBr
fluoresces under UV light

PCR detection methods – real time PCR/qPCR
• Various fluorescent chemistries have been designed to correlate
production of the PCR product to fluorescence intensity
• SYBR Green Dye
• Taqman® probes

Real time PCR – advantages & disadvantages
• Rapid – no post-reaction processing is
necessary
• Increased sensitivity - can detect smaller
amounts of starting material than traditional
PCR
• Quantitative rather than qualitative
• Large dynamic range
• Significantly greater sample throughput
• Requires expensive instrumentation

Real time PCR – data analysis
• The PCR reaction can be divided in to four phases
• Linear ground phase – fluorescence is below
background levels, baseline fluorescence is calculated
• Early exponential phase – fluorescence crosses a
defined threshold, the cycle at which this occurs is
known as Ct
• Exponential phase – optimal amplification
• Plateau phase – reagents become limiting, the
reaction slows down

• Instrumentation is required for analysis of real time PCR data
Real time PCR – data analysis

Real time PCR - instrumentation
• A wide range of instrumentation is commercially available
• Factors to consider when selecting a reader include:
• Price
• Size
• Block format
• Well capacity
• Individually programmed wells
• Heated lids
• Maximum ramp rate / run time
• Software integration......

Immuno-PCR
ELISA
• Adaptable to the detection of any protein
• Not adequate for the detection of low
abundance analytes
Real time PCR
• Provides exponential signal amplification
• Cannot be used directly for antigen
detection
Immuno-PCR

Immuno-PCR – main assay steps

Immuno-PCR – assay optimization
• Maximize the specific readout, minimize background signal
• Plate coating
• Plate washing
• Blocking
• Sample incubation
• Plate layout
• Choice of antibodies

Immuno-PCR – plate coating
• Antibody binding via non-specific adsorption
• Typically performed overnight at 4oC
• Carbonate, bicarbonate or borate buffer at pH>9
• Optimal concentration of the coating antibody should be
determined
• Starting range of 0.2 - 20µg/ml
• Grid layout for simultaneous testing of multiple antibody
concentrations

Immuno-PCR – plate washing
• Wash steps are essential for the removal of unbound
reagents
• Typical wash buffers include Phosphate Buffered Saline
(PBS) and Tris Buffered Saline (TBS)
• A low concentration of detergent (0.01-0.1% v/v) is often
added
• The number and duration of washes should be optimized

Immuno-PCR – blocking
• Blocking is critical to prevent non-specific binding
• Maximizes signal: background (S:B) ratio
• The blocking buffer is typically a weak solution (1%-5%
w/v) of protein diluted in wash buffer
• Bovine Serum Albumin (BSA)
• Non-fat dried milk
• Gelatin
• Casein
• Whole serum
• An overnight blocking step can improve S:B

Immuno-PCR – sample incubation
• Dilution of test samples can reduce background effects
caused by the nature of the sample material
• Short incubation times are often sufficient for antibody:
antigen binding
• Incubation temperature should be optimized

Immuno-PCR – plate layout
• Positive controls – spiked with known concentrations of antigen
• Negative controls – sample material without antigen
• Calibration curve
• Used to calculate the antigen concentration of test samples
• 10-fold serial dilutions, or lower, of the antigen
• All samples should be analyzed in duplicate
• Controls and standards should be diluted in the same matrix as the
test samples

Immuno-PCR – choice of antibodies
• Requires highly specific antibodies
• Recognition of a common molecular motif will increase
background signal
• Capture and detection antibodies should each target a
different epitope
Polyclonal Monoclonal
• Subject to variability between different
immunized animals
• A permanent antibody supply cannot be
guaranteed
• Immunogen affinity purification is
required
• Large quantities of a specific antibody,
recognizing a single antigenic epitope
• Uniform performance
• Immunogen affinity purification is not
necessary

Immuno-PCR – advantages & disadvantages
• Provides exponential signal amplification
• Extremely low limit of detection (pg - fg)
• Suitable for small sample volumes
• Compatible with complex samples
• Fewer incubation steps than an ELISA, improved
assay reproducibility
• Rapid time to results
• Wider dynamic range than an ELISA
• Amenable to multiplexing
• Requires conjugation of an
antibody to an oligonucleotide

ThunderLink® PLUS
• An easy-to-use kit for the production of
stable antibody-oligonucleotide conjugates
• No specialist knowledge required
• 30 minutes activation, 60 minutes
conjugation
• Optional clean-up step to remove unbound
oligo

ThunderLink® PLUS - advantages
Advantages
• Quick and easy to use
• High levels of antibody and oligo recovery
• Covalent bond
• Unidirectional chemistry
• Use your own oligo and antibody, at your desired ratio
• Linking chemistry works at both the 5’ and the 3’ end
• Optional post-conjugation clean-up step for removal of unbound oligo
• Positive control antibody and oligo included to confirm conjugation success
• Freeze dried
• Stringently QC tested

ThunderLink® PLUS – antibody considerations
• Activation of 100µg antibody in 100µl suitable
buffer
• Purified antibody, 1mg/ml
• AbSelect™ antibody purification kits
• Buffer exchange
• Concentration of the antibody
• Purification from TCS, ascites or serum
Buffer components Antibody buffer
pH 7-9
Amine free buffer ✓
Non-buffering salts (e.g. NaCl) ✓
Chelating agents (e.g. EDTA) ✓
Sugars ✓
Glycerol <50%
Thimerosal 
Merthiolate 
Sodium azide* <0.1%
BSA* <0.1%
Gelatin* <0.1%
Tris <20mM
Glycine 
Primary amines (e.g. amino acids) 
Thiols (e.g. mercaptoethanol, DTT) 
* Individually the concentrations shown should not affect the reaction, however
in combination with additional compounds that are not recommended above a
certain concentration the reaction may be affected.

ThunderLink® PLUS – oligo considerations
• Activation of 60-100µM oligo in 100µl suitable
buffer
• Must be HPLC purified
• Single-stranded oligos of 10-120 bases
• Terminal amine group
• Double-stranded oligos up to 80 base pairs
• Only one strand should be terminally aminated
• Linking chemistry works at either end of the
oligo
• Compatible with biotinylated or fluorescent dye-
conjugated oligos
Buffer components Oligonucleotide buffer
pH 6-8
Amine free buffer ✓
Non-buffering salts (e.g. NaCl) ✓
Chelating agents (e.g. EDTA) ✓
Sugars ✓
Glycerol <50%
Thimerosal 
Merthiolate 
Sodium azide* <0.1%
BSA* <0.1%
Gelatin* <0.1%
Tris 
Glycine 
Primary amines (e.g. amino acids) 
Thiols (e.g. mercaptoethanol, DTT) 
* Individually the concentrations shown should not affect the reaction, however
in combination with additional compounds that are not recommended above a
certain concentration the reaction may be affected.

ThunderLink® PLUS – antibody: oligo ratio
• The antibody: oligo ratio can easily be varied
• The antibody: oligo ratio is an average since a population of labeled antibodies will
be produced
• The conjugation efficiency is typically 95-100%
Volume of activated
antibody (µl)
Volume of activated
oligo (µl)
Volume of wash buffer
(µl)
Antibody: oligo ratio
300 300 0 1:15
300 200 100 1:10
300 100 200 1:5
300 60 240 1:3
300 20 280 1:1

Custom services
• Antibody-oligo micro-optimization service
• Six conjugates – different chemistries, different antibody: oligo ratios
• Custom antibody-oligo conjugation service
• Production of chosen conjugate at required scale
• Both services include
• Antibody clean-up prior to conjugation
• Removal of unbound oligo post-conjugation
• QC gel to demonstrate successful conjugation

Summary
• Immuno-PCR combines the specificity of an ELISA with the sensitivity
of PCR
• Rapid generation of robust and reproducible data
• Amenable to multiplexing
• Thunder-Link® PLUS for quick and easy conjugation of antibodies to
oligonucleotides
Thank you!

Thank you!

Get in touch..
For more information please get in touch or visit our website
+44 (0) 1223 661000
www.linkedin.com/company/innova-biosciences@InnovaBioSciwww.facebook.com/InnovaBiosciences

A Guide to Immuno-PCR

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