selection, validation and optimization of a new...
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Selection, validation and
optimization of a new antibodyHazel Chambers-Smith
Sri Lanka Workshop on Basics of Immunohistochemistry, June 2019
Where to start?• So, someone has been to a conference and
now we “have to get this new antibody”
• Need to ask:
• Is it relevant to our patients?
• Are there journal articles to support the
use?
• Is there a published method?
• Known positive and negative targets?
• Can it be used on paraffin embedded
tissue?
• Is there a distributor for this product in my
area???
Which one to choose?
• For any given target antigen, there
could be 100 primary antibodies which
claim to detect this target
• Key is for the antibody to have a high
sensitivity and high specificity
• And ideally proven to have good results
with your IHC platform
Specificity
• Ability of the antibody to bind selectively
to a single epitope on an antigen
Sensitivity
• The relative amount of antigen that an
IHC technique is able to detect
IHC program software
• Usually piggy back
from an existing
protocol
• Common to just
change the retrieval
and Ab incubation
time
• + amp for difficult
antibodies
• Monoclonal antibodies are preferred
because of their higher affinity for a
given epitope
• Mouse monoclonals are traditionally
available
• Rabbit monoclonals are gaining
following due to the rabbit antibodies
having an improved recognition for
human antigens.
Check the clones
• Log on to NORDIQC
https://www.nordiqc.org/
• Check previous runs to
determine which
antibody to use
• Gives great information
on preferred retrieval,
detection and platforms
• To consider:
• Don’t wait for a failed external/internal
QA
• NORDIQC can help with choosing to
replace a current antibody if not staining
to specifications
• Implementation and validation steps will
be the same.
Choosing the right clone is
important!
• ALK antibody
• LHS: RHS:
D5F3 clone(for Lung) ALK1 (for ALCL)
The process of optimization
• Conditions for testing of control tissue
for validation should be the same as the
patient tissue you will test
• Fixation, processing, sectioning etc
should be standardised for control
tissue
• Use of a tissue microarray can be useful
but is time consuming to prepare
Controls
• Correct controls essential to
demonstrate calibrated staining
• Nordic QC have great control
information for common antibodies
• Data sheets/journal articles informative
for your rarer antibodies
• Low expressors best to calibrate
antibodies and demonstrate sensitivity
Oestrogen Receptor
Why use IVD antibodies?
• Suitable for in vitro diagnostic work
(routine testing)
• Responsibility for validation and application
of antibody sits with the vendor
• Start with the suggested dilution from the
vendor
• Use correct antigen retrieval (if indicated)
• Use known positive and negative tissues
• Review with known results (sometimes best
to have sent away some cases to a lab
routinely running the test so there is a
comparison)
• Document outcomes – keep a log of cases
stained
• Get sign off from a pathologist and scientist
RUO
• Research use only
• Responsibility for the
validation and
documentation of
sensitivity and
sensitivity lies with the
diagnostic lab
• Always buy IHC(P) if
you can
• Need to determine appropriate dilution
• Need to ascertain antibody range
• Compare background to visualisation of antigens
of interest
• Might be necessary to test a few different clones
against the antigen of interest
Every variable will affect the
quality of the stain
• Pre-analytical factors
• The antibody
• The diluent
• The retrieval buffer
• The detection kit
• The platform itself
• Calibration is essential!!!
Antigen retrieval
• Use a range of pH, temperature and
times to determine best range
Amplification
Sign off
• Once the antibody dilution, retrieval
method and staining method has been
validated, each step of the process
must be documented and maintained
• Record appropriate positive and
negative control tissues.
• Validation records for new antibodies
implemented must be available for
review at accreditation
Validation tips
• Make your document very simple
• I suggest a simple one page document
containing a table of cases used
containing type of expression
• A comparison with duplicates stained at
another lab
• Or confirmed molecular result if
applicable eg BRAF (on brain tumours)
BRAF at RCH
Correct optimisation will take you
from this
To this
In summary
• Enrol in appropriate quality assurance
program or inter-lab slide referral
• NordiQC
• UKNEQAS
• RCPAQAP(Australia-I am an assessor
)
• Review clones appropriate for use on a
regular basis
• Act on feedback from pathologists
Any questions?