The Importance of Antibody Validation


 As a researcher in the life sciences, antibodies are your main tools used in experiments. Reliable and reproducible data can be generated when high-quality  primary antibodies are properly used.  To be of “high quality” antibodies should be validated by methods that demonstrate the following antibody properties:

  • Sensitivity – “on target” binding properties
  • Specificity – “off target” binding properties  
  • Reproducibility – produced to ensure lot-to-lot consistency. 

 

A recently published report of the International Working Group on Antibody Validation (IWGAV) titled A Proposal for Validation of Antibodies established five conceptual pillars for assay specific antibody validation.  Rockland Immunochemicals supports this concept and is actively working with others to establish guidelines for antibody validation that can be applied globally. More reports on antibody quality and validation.

 

 Our highest priority has always been the  quality and performance of antibodies produced by Rockland.  By trusting your experiments to us, your data will reflect increased antibody specificity, low background staining, increased assay sensitivity, reduced incubation times and you will see consistent results from lot-to-lot.

 



 Reproducibility Issues in Life Science Research: Antibody Validation Challenges 

 

In this Biocompare documentary, researchers discuss what is being done in their labs to improve reproducibility, what’s being done by the manufacturers to decrease lot-to-lot variation, what’s being demanded by journals and funding agencies to ensure that certain standards are adhered to, and finally what role organizations and societies are playing to disseminate information and increase awareness of the problem:
 
   

 
Related content: Click here to read Biocompare's Q&A with Rockland's CSO, Carl Ascoli, Ph.D.



 Transparency, shared responsibility and partnership are essential elements of any plan to standardize antibody validation if the goal is to affect researchers worldwide. A 2017 article in Biocompare titled The Antibody Crisis: An Ongoing Discussion engaged thought leaders from around the globe including Rockland's CSO, Dr. Carl Ascoli who stated, “It is important for transparency that the nature of the immunogen, how an antibody was screened during development and specific details pertaining to lot-specific validation are communicated." Appropriately transparent data should be provided by the manufacturer to the user to allow replication of data and to gain confidence in antibody performance.    
 Biocompare1

 

 

 


We propose some basic guidelines for how researchers use antibodies. Key questions must be answered by researchers on the intended outcome of antibody based experiments. These issues should be resolved before experimental work commences, ideally before an antibody is produced or purchased. If these guidelines are followed the likelihood of obtaining valuable, reproducible data dramatically increases. 

 

 Validation-Guide  

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 gbsi 

 

The Global Biological Standards Institute (GBSI) and The Antibody Society recently organized the Antibody Validation: Standards, Policies, and Practices workshop that included more than 100 key stakeholders from academia, antibody producers, pharma, funders, and journals to share perspectives and contribute to tangible solutions for validating antibodies. A workshop report was issued by GBSI that summarized the conceptual framework agreed upon at the meeting for affecting change including a call for detailed assay-specific antibody validation standards.

 

 

 profile-carl   Rockland's CSO, Dr. Carl Ascoli spoke as a panelist during the Producers and Service Providers in a Pivotal Role: QC/QA and Certification discussion. 

 

 


Who should validate antibodies?

 

All manufacturers should validate all antibodies by at least a minimum criteria. Antibody validation data should be lot specific and the data should be communicated to the researcher in the form of a datasheet or certificate of analysis, which would accompany each unit of product shipped to the researcher. Data should demonstrate transparency and include information regarding the nature of the immunogen, how the antibody was screened during development and the final release validation data. It should also include properties like appropriate dilutions for use.

 

Validation data should be lot specific and assay specific. Researchers should carefully review provided information and, when necessary, independently validate an antibody before starting with research to accommodate any changes in buffers used, equipment or other parameters specific to your laboratory.

 

 

Application

 

 Antibody Application   Rockland scientists validate the performance of antibodies by assays commonly used by most researchers including ELISA, western blotting and immunohistochemistry.

Many Rockland products are validated in a variety of assays and are guaranteed to work as per the applications stated on the data sheet. 

 


Cross-Reactivity

 

  Antibody Cross-Reactivity   Rockland antibodies are tested at every stage of production using positive and negative controls to ensure the antibody targets the right protein. 

Rockland quality control standards require the highest possible specificity for antibodies. Reactivity to other species is listed on the data sheet.

 


Reproducibility


 Antibody Variability   Rockland manufacturing processes deliver unsurpassed repeatability in antibody production and minimal lot-to-lot variation.

Rockland adheres to QSR/cGMP quality standards (CFR: 21H part 820) with full reporting and traceability. A product data sheet is provided with each order.

  


 

Assay-specific Methods for Validating Antibodies

 

 Validating an antibody is not the same as characterizing an antibody.  A data image of an antibody used in an immunoassay with or without controls could be described as characterizing an antibody.  But validating an antibody means that you have determined the antibody’s sensitivity for the target, its specificity for off target binding and its reproducibility, especially to demonstrate that the antibody’s properties are consistent from lot-to-lot.

Legitimate methods for validating antibodies are immunoassay specific, meaning that the methods used for Western blotting would not necessarily apply to other applications such as ELISA, immunoprecipitation or immunohistochemistry. 


 

Selected Readings: Antibody Quality and Validation

 

 

1. Baker, M. Blame it on the Antibodies.  Nature 521:274-276.  May 2015. 

 

2. Begley, C.G. and Ellis, L.E.  2012.  Raise standards for preclinical cancer research.  Nature 483; 531-533.

 

3. Birabaharan, J. and Ascoli, C.A.  The Antibody Dilemma:  Shortcuts Taken by Antibody manufacturers and End-Users have Led to a Reproducibility Crisis.  Article in Genetic Engineering and Biotechnology, August 2015.

 

4. Bordeaux, J., Welsh, A.W., Agarwal, S., Killiam, E., Baquero, M.T., Hanna, J.A., Anagnostou, V.K, and Rimm, D.L. Antibody ValidationBiotechniques. 2010 Mar; 48(3): 197–209.

  

 5. Bradbury, A. and Plückthun, A.  2015.  Standardize antibodies used in research.  Nature 518; 27-29.

  

6. L.P. Freedman, M.C. Gibson, A.R.M. Bradbury, A.M. Buchberg, D. Davis, M.P. Dolled-Filhart, F. Lund-Johansen and D.L. Rimm, 2016.  The need for improved education and training in research antibody usage and validation practices. BioTechniques 61;1:6–18.

 

7. D. Shastri.  Survey finds laissez-faire attitude towards validating antibodies. Article in Science, July, 2016.

  

 8. R.D. Polakiewicz.  Antibodies: the Solution is Validation.  Nature 518:483.  February 2015.

 

 9. M. Uhlen, A. Bandrowski, S. Carr, A. Edwards, J. Ellenberg, E. Lundberg, D. L. Rimm, H. Rodriguez, T. Hiltke, M. Snyder, T. Yamamoto.  A proposal for Antibody ValidationNature  Methods 13:10; 823-827.

 

10. Weller, M.G. 2016.  Quality Issues of Research Antibodies.  Analytical Chemistry Insights 11; 21-27

 


 

Validating Antibodies by Western Blotting

 

 

 Legitimate Methods Western Blotting Validation

 

1. Using purified proteins.

 

2. Using lysates overexpressing the target protein (ectopic expression).

 

3. Using native cells or tissue.

 

4. Special Considerations for Antibodies that Recognize Post Translational Modifications.

 

 


 

1. Purified Proteins, native or recombinant, can be used to assess reactivity with a specific antibody. This method has limited value but is a legitimate method for antibody validation if other options are not available. The immunogen or a protein produced by immunoprecipitation (IP) may also be used. Size markers and positive and negative controls are required for each Western blot.

 

 Options that add confidence to the assessment:

 

a. Add varying amounts of protein loaded to different lanes of the gel to assess antibody reactivity.

 

b. If recombinant, a second antibody against the fusion tag, such as anti-GFP or anti-FLAG could be used to co-localize with the target protein.

 

c. If recombinant, a second protein with the same promoter and fusion tag could be used to demonstrate that the antibody does not identify the tag portion which would demonstrate a lack of specificity.

 

d. If recombinant, a null or mock unpurified lysate could be used to demonstrate that the antibody does not identify non-specific bands which would demonstrate a lack of specificity.

 

e. Combine a. and d.  Spike a null lysate with varying amounts of purified protein and load into different lanes of the gel to show band intensity increases when the amount of protein is increased and that the antibody does not react non-specifically.

 

f. Multiple antibodies against the target could be used to co-localize with the target protein.

 

 g. The target protein could be analyzed by mass spectrometry for proper identification.

 


 

2. Lysates from cells overexpressing the target protein,  either cell-free expression systems like rabbit reticulocyte lysates, or stably or transiently transfected cell lysates can be used to assess reactivity with a specific antibody. This method has greater value than using purified protein but is not as valuable as detecting native protein in cells or tissues. Size markers and positive and negative controls are required for each Western blot.    

 

 Options that add confidence to the assessment:

 

a. Options 1.b-f specified above for purified proteins may also apply to lysates with minor adjustments incorporated into the test material as needed.

 

b. Induce varying degrees of protein expression to produce different amounts of protein loaded to different lanes of the gel to show band intensity increases when expression levels increase, e.g. vary amount of ligand that controls expression by binding to promoter.

 

c. Spike a null lysate with varying amounts of purified protein and load into different lanes of the gel to show band intensity increases when the amount of protein is increased.

 

d. Use lysates that express the target protein at a level anywhere from 0.2X to 5X of native expression levels as determined by the literature or by measuring native RNA or protein.

 

e. Two-dimensional Western blotting is used to identify the target protein by both size and isoelectric point.

 

f. The ability of the antibody to bind processed forms, orthologs and isoforms is assessed.

 

 Results that diminish confidence in the assessment:

 

g. A significant number of major or minor bands are observed in lysates that do not correspond with known forms of the target protein.


h. Additional band(s) are observed near the molecular weight of the target protein
 

 

3. Lysates from native cells expressing the target proteineither cultured cells or tissues that represent normal, disease state or drug induced/stimulated systems can be used to assess reactivity with a specific antibody. This method has the greatest value for assessing the performance of antibodies for research.  Size markers and positive and negative controls are required for each Western blot.    

 

 Options that add confidence to the assessment:

 

a. Options 2.e-f specified above for lysates from cells overexpressing the target protein would apply when testing lysates from cells expressing native protein. 

 

b. Multi-lysate panels could be produced from cells known to express or not express the target of interest based on genomic or proteomic studies.  Antibody binding would correlate with known expression levels.

 

c. The levels of target protein can be modulated by adding drugs, growth factors or by altering the growth conditions of the cells which would result in different amounts of protein.  These samples should show corresponding changes in band intensity when loaded to different lanes of the gel.

 

d. Genetic manipulations could be used to affect the expression of the protein such as knock out genes, siRNA, shRNA, CRISPR/Cas 9 and other approaches of RNA Interference and gene manipulation.  Controls showing proper expression levels are required for comparison.

 

 Results that diminish confidence in the assessment:

 

e. A significant number of major or minor bands are observed in lysates that do not correspond with known forms of the target protein.

 

f. Additional band(s) are observed near the molecular weight of the target protein.

 


 

4. Special considerations for antibodies that recognize   Post-Translational Modifications (PTMs) are helpful when the target protein has been specifically modified with a PTM that can be regulated (e.g., phosphorylation, acetylation, or glycosylation). PTM specific antibodies are often produced using modified and unmodified peptides, so peptide conjugates are considered to be legitimate tools for validation, e.g. validate using peptide BSA conjugates when pepti de KLH conjugates are used for immunization. Size markers and positive and negative controls are required for each Western blot.    

 

 Options that add confidence to the assessment:

 

a. Perform a peptide competition assay where the PTM specific antibody is pre-incubated with modified peptide and/or unmodified peptide.  Pre-incubation with the modified peptide should eliminate PTM specific antibody binding and diminish or eliminate staining of the target protein.   Pre-incubation with the unmodified peptide should have no effect on PTM specific antibody binding. 

 

b. Use a peptide array to confirm the reactivity of the PTM specific antibody for the modification in context with surrounding amino acids.  Typically the array will include other protein modifications and similar amino acid substitutions to confirm specificity.  Peptide arrays are often used to assess the specificity of epigenetic modifications of histones.  

 

c. Similar to option 3.c., use an inducing agent to activate the PTM and enhance staining of the target protein.  Conversely, staining of the target protein should be absent or diminished when an inhibitory agent is added.

 

d. Perform in vitro dephosphorylation/deacetylation of a phosphorylated/ acetylated recombinant protein by enzymatically treating the protein with a phosphatase/deactylase.   The staining of the target protein should be absent or diminished upon phosphatase/deactylase treatment.

 

e. Perform in vitro phosphorylation/acetylation of an unphosphorylated/unacetylated recombinant protein by enzymatically treating the protein with a kinase/acetylase.   The staining of the target protein should be present or enhanced upon kinase/acetylase treatment.

 

f. Alter the recombinant protein by introducing a point mutation, such as a transversion to change serine to alanine, e.g. a codon change from TCA to GCA.  This will block phosphorylation of the target protein.  The staining of the target protein should be absent upon introduction of the mutation.

 

 Results that diminish confidence in the assessment:

 

g. A significant number of major or minor bands are observed in lysates that do not correspond with known forms of the target protein.


h. Additional band(s) are observed near the molecular weight of the target protein.

 

 


     
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