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What are Validated Antibodies? 

 

Dear Doctor  

 

In this issue of Biospeak, we focus on the critical issue of Antibody Validation.

 

Increasing concern is being expressed in various forums about antibody quality and this has led to the concept of 'Validated Antibodies'. At Biosensis we are pleased to see this increasing scrutiny as research suffers when poor quality reagents are used.

 

We first highlighted this issue 5 years ago (Antibody Quality). Since then, several initiatives have added weight to address the issue with journals rejecting manuscripts that fail to provide sufficient detail of antibody generation and specificity, i.e., validation (see for example Saper: J Comp Neurol; Couchman: J Histochem Cytochem).

 

Many publications continue to address this issue (see Howat et al: Methods; O'Hurley et al: Molecular Oncology). In addition, a frustrated research community is seeking ways to create databases of validated antibodies and banks of new antibodies certified by consortia of scientists (see Perkel: BioTechniques). Some even argue to throw away all polyclonal antibodies and monoclonal antibodies and use only recombinantly generated antibody derivatives produced by a $US 2 Billion public fund (Bradbury et al: Nature).

 

So just what is a 'Validated' Antibody? While not easy to define, here are 3 examples (Old, Recent, New) taken from our catalogue that show how complex the issue can be.

A few Validated Antibodies from Biosensis
(M-006-100) - MC192
(M-009-100) - MLR2
(M-1586-100) - MOAB-2
New MOAB-2 antibody
MOAB-2 Image 1
MOAB-2 is a new highly specific amyloid beta (Aβ) monoclonal antibody used for Alzheimer's research. A large amount of validation data has been generated for its use in Western blot, IHC and sandwich ELISA for oligomeric Abeta. (Catalog No: M-1586-100).
 

OLD (Seasoned!) 

MC192 (M-006-100). A monoclonal antibody generated by researchers in the 1980s and cited in more than 250 publications. It reacts against the rat form of p75NTR but not human, mouse or other species. Validated for western blot and immunohistochemistry. However, several publications have used the antibody as an antagonist agent when the initial and many subsequent publications (and our datasheet) have clearly demonstrated this antibody causes increased binding of NGF to the p75NTR receptor.   

M-006-100 Immunofluorescent detection of p75NTR in cultured rat dorsal root ganglion (DRG). (A) Detection of p75NTR using mouse monoclonal antibody to rat p75NTR [MC192] M-006-100. (B) Detection of betaIII-Tubulin. (C) DAPI staining. (D) Merged A, B and C.   

 

 

 

 

 

M-006-100 Immunohistochemical staining of p75NTR in rat motor neuron on lesioned sciatic nerve using mouse monoclonal antibody to rat p75NTR [MC192], catalogue number M-006-100.

 

 

 

 

 

 

M-006-100 Sample: PC12 cell lysate in RIPA buffer with protease inhibitors; SDS-PAGE: Denaturing (10 min @ 70�C), non-reducing; Electrophoresis: 4-20% Bis-Tris; Transfer: Tris-Glycine buffer, nitrocellulose membrane (0.45 μm); Blocking: 5% skim milk in TBST, 1 hour at RT; Primary antibody: 5 and 10 μg/mL MC192 in blocking buffer, overnight incubation; Secondary antibody: Anti-mouse-HRP (1:6000 dilution); Detection: Chemiluminiscence; Predicted MW of rat p75 based on amino acid sequence: 43 kDa; Observed MW due to post-translational modifications: 60 kDa.

 

   

 

YOUNG (Adolescent!)   

MLR2 (M-009-100). (see also BEK-2219-2P; BEK-2220-2P) A monoclonal antibody generated more recently by researchers using knockout mice. The immunization protocol was designed to generate antibodies that would be useful across many species such as human, mouse and rat. Despite having only 14 citations, it has strong validation data showing usefulness in many techniques including FACS, Western blot (for native protein only), IHC, immunopanning, sandwich ELISA (when paired with another p75NTR antibody), immunoprecipitation and in vivo targeting for gene delivery.

   

M-009-100

Flow cytometry analysis of endogenously expressed p75NTR on ShSY5Y cells. X-63 conjugated control IgG represents negative control for all experiments.

  

 

 

 

 

 

M-009-100 Immunofluorescent detection of p75NTR in cultured mouse NSC34 cells using a mouse monoclonal antibody to human p75NTR [MLR2], catalogue number M-009-100.

 

 

 

 

 

 

 

 

M-009-100 Staining of transverse sections of E9.5 mouse embyros fixed in 4% paraformaldehyde in PBS and immunolabelled with anti-p75 antibody MLR2 (M-009-100, 1:200, green), anti-cleaved-caspase-3 (red, upper panel) and anti-phospho Histone H3 antibodies (PHH3, red, lower panel). Primary antibodies were incubated at 4�C overnight. Scale bar = 200 μm. Courtesy of Dr S. Wiszniak, SA Pathology.

NEW (Embryonic!)    

MOAB-2 (M-1586-100). (see also BEK-2215-2P) A new antibody less than 3 years old, so only used in 10 publications to date. However, the researchers who produced this novel Abeta antibody and Biosensis R&D scientists have generated a large amount of validation data for its use in Western blot, IHC and sandwich ELISA for oligomeric Abeta.  

 

M-1586-100 Sample combined cortex and hippocampus from 12 month old 5xFAD mice. 1.25-25 mg of FA extraction were analyzed using the standard Western blot procedure as described in Youmans, Molecular Neurodegeneration, 2012. MOAB-2 was used at a concentration of 1ug/ml.

 

 

 

 

 

 

 

 

M-1586-100 Immunohistochemical detection of Aβ and APP in coronal sections of the frontal cortex of 1-month old 5xFAD mice. Sections were co-stained with M-1586-100 or APP antibodies (N-terminus and C-terminus). MOAB-2 staining was punctate and did not co-localize with either APP antibodies. Figure courtesy of Youmans et al (2012).

 

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Should your manuscript ever be rejected by ANY journal because of insufficient information about a Biosensis antibody, we will gladly refund TWICE the purchase price of that antibody!


By researchers for researchers!   

The staff at Biosensis have over 60 years of combined experience in manufacturing and working with antibodies and have published over 340 peer reviewed research publications in the field of neuroscience. We guarantee to provide you with the antibody information you need to get your research published.

 

If you have a query on any of our products, one of our experienced researchers can help at biospeak@biosensis.com.
   
Until next time, good luck with your research.
 
Sincerely,
Tina
 
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