Antibody Validation:

Discovery Demands It

•A dream you have will come true with

antibodies.

•A very attractive person has a message for

you

about antibodies.

•Nothing astonishes so much as common

sense and

plain dealing with antibodies.

•Integrity is the essence of everything

successful

with antibodies

•To be old and wise, you must first be young

and

stupid with antibodies.

•The most important thing in communication is

to

hear what isn’t being said about antibodies.

From Prophecy To Validation

A Few More

Customers are expecting an antibody to work the first and every

time used

Customers are seeking proof of antibody quality

A majority of commercially available antibodies lack sufficient

information to enable an informed choice by the customer

Even where data is available, lot-to-lot testing is often not

performed, leading to the purchase of unqualified antibodies

Consequently, customers spend time and precious sample

validating and optimizing commercial antibodies – time that should

be used on their research efforts

Expectations of Antibody Consumers

Customer pain points with research antibodies

Poor quality accounts for >50% of

pain

Specificity – proper controls and models to ensure specificity

Sensitivity – detects endogenous levels of antigen

Reproducibility – reproducible within and between lots

Biological Relevance – tested in the correct model

system under the correct conditions

Some Principles Behind Good Antibody Validation

Validated Western Blotting Methodology

Example method; cell/tissue lysis

Two or more verified cell lines or tissue lysates run

Defined controls to ensure specificity, sensitivity, and reproducibility

Defined lysis and western protocols

Thorough antibody optimization and data preparation

Additional validation including IF, IHC, FC, IP and ChIP

Western blotting highlights

• MW of band should not deviate +20% or -10% of estimated MW, in presence of two controls: positive

(unpurified sera) and negative (naïve sera).

• Positive deviation (observed is heavier than expected) must be justified by PTMs

• Under-shifts due to truncated isoforms, and isoforms must cross-react with the immunogen sequence

• Estimated MW, PTMs, and isoforms must be obtained/confirmed via UniProt (UP)

• Peptide blocking with 3 increasing concentrations

• Bands are blocked gradually and the band at highest concentration disappears

• The species of cell lines/tissues used is identical to the target protein

• Positive in minimum of two cell lines/tissues

• Number of non-specific bands is less than 2, and weaker than specific band

• Test minimum of 2 dilutions (1:500, 1000) for antibody

• Embedded MW marker system must be deployed and used in conjunction with each approved test -

selected products

Western Blot Standards

There Are Consequences For Quality

Short-Term and Long-Term

• Customer Satisfaction

• Market Position

• Resource Allocation

• Scientific Impact

Product Pipeline Database (PPD) – Image Viewer

D1 Dopamine Receptor

AntibodyUBE2K Antibody

SPHK1 Antibody

Validation Data – Western Blot

Validation Data - Flow

G1=R1control

Human peripheral blood

lymphocytes stained with

purified OKT3, followed by

anti-mouse IgGs Alexa Fluor

488.

control G1=R1

R1

R2

Human peripheral blood

lymphocytes stained with

purified 4B2, followed by

anti-mouse IgGs Alexa Fluor

488.

R1: Human peripheral

blood lymphocytes

R2: Human peripheral

blood monocytes

Validation Data - Immunohistochemistry

Validation Data - Immunofluorescence

High throughput Western Blot Platform

Mature IF/IHC/FC Platform

Fluorescence microscope Flow cytometry instrumentHigh throughput –Flow cytometry instrument

Tissue slicing machine Paraffin embedding machine Microscope

Specialized Cell Room

Professional technical tea

m

Crowd Sourcing Validation – A Success Story

Specialty Meetings

Validation Leaders

Educational Materials

Lab Reach Out

Data

Accelerating Scientific Impact

Validation Partnering

Autophagy

Phosphorylation

Targets

Validation Leaders Publications

ISO9001:2008 (Certificate number

CN07/00676)

Suzhou animal facility is fully

accredited by AAALAC

International (Unit number 1326)

Lab Certifications:

The Validation of Validation

Discovery Demands It

“To know even one

life has breathed

easier because you

have lived. This is

the meaning of

success.”

- Ralph Waldo

Emerson