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?