Antibody validation

The usefulness of antibodies in different assays is dependent on both sensitivity and specificity of epitope binding, and in order to provide the best estimate of protein expression across tissues and cells, antibody validation is a crucial part of the Human Protein Atlas. All antibodies are validated by a set of defined criteria, as described below. Only antibodies that pass the minimum criteria of standard antibody validation are published on the Human Protein Atlas. In addition to the standard quality assurance, enhanced antibody validation strategies are performed in an application-specific manner. The different criteria for enhanced antibody validation are described below.

Standard antibody validation

All antibodies produced internally within the Human Protein Atlas project (HPA antibodies) must pass steps 1-4 in the list below in order to be used for immunohistochemistry and immunocytochemistry/IF. Steps 5-7 provide the basis for evaluating and scoring the antibody reliability. All antibodies that provide a reasonable pattern of immunoreactivity are added to the Human Protein Atlas portal. Feedback from the research community is appreciated and needed for continuous curation of data.
Quality assurance steps for antibodies generated within the Human Protein Atlas project:

1. The antigen (protein epitope signature tag (PrEST)) for a protein is selected as a stretch of 20-150 amino acids with as low identity as possible to proteins from all other putative protein-coding genes, and not including signal peptides or transmembrane regions. Multitarget PrESTs are PrESTs that have more than 80% identity to proteins from more than one gene, and are expected to generate antibodies with multiple targets.
2. Plasmid inserts are sequenced to assure that the correct PrEST sequence is cloned.
3. Size of the resulting recombinant protein (including the specific PrEST) is analyzed using mass spectrometry to assure that the correct antigen has been produced and purified.
4. To control for cross-reactivity, affinity purified antibodies are tested for sensitivity and specificity on protein arrays consisting of glass slides with spotted PrEST fragments.
5. Antibody specificity is analyzed using Western blot in a standardized setup. Total protein lysates from a limited number of tissues (liver and tonsil), cell lines (RT4 and U-251 MG), and human plasma are used to evaluate the antibody target binding in a Western blot setting. Antibodies with an uncertain standard Western blot are reanalyzed using an over-expression lysate as a positive control.
6. Immunohistochemical staining of normal and cancer tissue is examined and annotated by specially educated personnel, and the staining patterns are compared with available gene/RNA/protein characterization data.
7. High resolution confocal microscopy images of human cell lines stained by indirect immunofluorescence are annotated for subcellular localizations by trained cell biologists, and the subcellular localization patterns are compared with the immunohistochemical staining and available experimental protein characterization data.

For antibodies supplied through commercial or other academic sources (CAB antibodies), immunocytochemistry and immunohistochemistry have been performed and validated in a similar manner as for HPA antibodies. These antibodies have also been tested on Western blot in a standardized setup. For each commercially available antibody, a link to the antibody provider is given on the "Antibody validation" page. For further validation we refer to quality controls provided by the respective company. Detailed descriptions of the strategies used for standard antibody validation in the different assays are available further down on this page.

Enhanced antibody validation

Antibodies used for Western blot, immunocytochemistry and immunohistochemistry in the Human Protein Atlas undergo enhanced antibody validation based on the five "pillars" described by the International Working Group for Antibody Validation (IWGAV), presented in "A proposal for validation of antibodies" (Uhlen M et al, 2016). The enhanced validation principles are adapted for validation in Western blot, immunocytochemistry and immunohistochemistry applications. Antibodies that fulfil the criteria are labelled "Enhanced". The following Enhanced antibody validation strategies are used for each assay:

• Immunocytochemistry: Genetic validation, Recombinant expression validation, and Independent antibody validation
• Immunohistochemistry: Orthogonal validation and Independent antibody validation
• Western blot: Genetic validation, Recombinant expression validation, Independent antibody validation, Orthogonal validation, Capture MS validation

Detailed descriptions are available under each section describing the different assays.

Immunocytochemistry - cells

Standard antibody validation - ICC

For each antibody, the observed staining in the different cell lines is assigned a validation score based on concordance with available experimental gene/protein characterization data in the UniProtKB/Swiss-Prot database. The validation scores for up to three cell lines are merged into one of the main categories; Supported, Approved, or Uncertain, to represent the overall antibody staining in all analyzed cell lines.

Validation scores for Immunocytochemistry/IF:

Supported

• The observed location(s) is(are) supported by experimental gene/protein characterization data (UniProtKB/Swiss-Prot).

Approved

• One/multiple location(s) with no available experimental gene/protein characterization data.
• One/multiple location(s) where experimental gene/protein characterization data is partly supporting and partly conflicting.

Uncertain

• Location not consistent with experimental gene/protein characterization data.

The validation of antibodies targeting PrESTs encoded by two or more genes (here called multitargeting) is based on the conformance of the expression pattern to available gene/protein characterization data. Similarity between paired antibodies is not taken in account due to the complexity of multiple gene targets.
Validation scores for Immunocytochemistry/IF - multitargeting antibodies:

Supported

• The multitargeting antibody yielding a staining pattern consistent with available gene/protein characterization data for all of the genes.
• The multitargeting antibody yields a staining pattern that is partly consistent with available gene/protein characterization data for all of the genes.

Approved

• The multitargeting antibody yields a staining pattern with no available gene/protein characterization data.
• The multitargeting antibody yields a staining pattern that is consistent with available gene/protein characterization data for at least one of the gene's but not all.

Uncertain

• The multitargeting antibody yields a staining pattern that is not consistent with available gene/protein characterization data.

Enhanced antibody validation - ICC

Genetic validation - siRNA

For an enhanced validation of the antibody and thereby confirming the determined subcellular localization of the target protein, the staining procedure is repeated on siRNA transfected U-2 OS cells in order to knock-down the expression of the protein (Stadler C et al, 2012).
A reverse solid phase transfection protocol is used to coat cell seeding surfaces with siRNA and transfection reagents prior to cell seeding. After siRNA transfection has occurred, cells are fixed and stained according to the standard protocol. For each antibody, the assay is performed in duplicates using two different siRNAs sources, and the results are compared to negative control cells transfected with scrambled siRNA.
Images are acquired using objectives with 10x- and 40x-magnification. An automated image analysis protocol segments the cells and extracts features from all acquired images before statistical software automatically compare the cell population median staining intensity between siRNA coated and negative control samples.
Relative Fluorescence Intensity (RFI) denotes the percentage of remaining staining intensity after siRNA-mediated down regulation. The distribution of RFI for the cells within a sample are presented in a box-plot and the significance of the down-regulation are evaluated using the Wilcoxon rank sum test (Mann-Whitney). A p-value below 0.01 is considered significant.
For each siRNA assay the decrease in antibody-based staining intensity upon target protein downregulation is evaluated.

Antibodies that meet one of the following criteria will receive the validation score "Enhanced" by genetic method:

• Significant downregulation > 25 % by both siRNAs.
• Significant downregulation > 25 % by one siRNA and > 10 % by the other.
• Significant downregulation > 25 % by one siRNA.

Recombinant expression validation - Tagged protein/GFP

Antibodies targeting a subset of genes are further analyzed in HeLa cell lines stably expressing GFP-tagged target protein.These cell lines are kindly provided by the group of Professor Anthony Hyman, Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany (Poser I et al, 2008; Skogs M et al, 2017). They are produced using a Bacterial Artificial Chromosomes (BAC) TransgeneOmics technology. BACs contain all regulatory elements and a transfection with BACs results in near-endogenous expression of the recombinant protein. Analysis is performed directly on the clone pool where individual cells show variations in tagged protein expression level. An anti-GFP antibody is used to enhance the signal in order to detect even low abundant tagged target protein.
All images are manually annotated to one or several subcellular locations. The location of the tagged protein is taken into account when performing the knowledge-based annotation of subcellular location for each gene described here.
The antibody staining intensity is classified as negative, weak, moderate or strong based on the laser power and detector gain settings used for image acquisition in combination with the visual appearance of the image. GFP intensity is classified as positive or negative.

For each GFP validation assay the signals of the antibody staining and the GFP-tagged protein is compared.

Antibodies that meet one of the following criteria will receive the validation score "Enhanced" by recombinant expression:

• Antibody staining overlaps with tagged protein.
• Antibody staining overlaps with tagged protein but shows additional locations.

Independent antibody validation

The protein localization(s) can be validated using independent antibody strategies. Two (or more) independent antibodies directed towards independent epitopes (non-overlapping) on the protein may be used to assess the reliability of the staining. Independent antibodies are scored based on their staining pattern similarity with its "sibling" antibodies.

Antibodies that meet the criteria of independent antibody validation will receive the validation score "Enhanced".

Immunohistochemistry - tissues

Standard antibody validation - IHC

For each antibody, the observed staining is assigned a validation score. The validation score is based on the result of two different validations that are separately evaluated: literature conformity and RNA consistency. Literature conformity refers to conformance of immunohistochemistry data from 44 normal tissues with available gene/protein characterization data in scientific literature and data from bioinformatic predictions. UniProt is used as the main source of gene/protein characterization data and when relevant, available publications and other sources of information are researched in depth. Extensive or sufficient gene/protein data requires that there is evidence of existence on the protein level and that a substantial proportion of published experimental data is available from literature and public databases. Limited protein/gene characterization data does not require evidence of existence on the protein level and refers to genes for which only bioinformatic predictions and scarce published experimental data is available. RNA consistency is based on a comparison of immunohistochemistry data from 44 normal tissues with internally and externally generated RNA-seq data.
The different levels of validation score are Supported, Approved or Uncertain.
Validation scores for immunohistochemistry using standard validation:

Supported

• Consistency with RNA-seq and/or protein/gene characterization data.

Approved

• Consistency with RNA-seq data in combination with inconsistency with, or lack of, protein/gene characterization data. Alternatively, consistency with protein/gene characterization data in combination with inconsistency with RNA-seq data.

Uncertain

• Inconsistency with, or lack of, RNA-seq and/or protein/gene characterization data.

Enhanced antibody validation - IHC

Orthogonal validation

Orthogonal validation is based on manual evaluation of the correlation between antibody staining intensity and corresponding mRNA levels across 37 normal tissues. For single-target antibodies where staining intensity and mRNA levels show the same pattern, two representative images corresponding to tissues with high and low expression of protein and mRNA are selected. If the difference in mRNA levels between the two representative tissues is evaluated as significant, the antibody will receive the validation score "Enhanced".

Independent antibody validation

This method is based on comparing the staining pattern using two single-target independent antibodies with non-overlapping epitopes. The spatial localization of the staining pattern generated by immunohistochemistry using the two antibodies is compared in 44 different normal tissues. For antibodies that show a similar spatial localization, four representative images are chosen for each antibody.

Antibodies that meet the criteria of independent antibody validation will receive the validation score "Enhanced".

Immunohistochemistry/IF - mouse brain

Standard antibody validation - IHC/IF

In order to generate and present reliable and valuable data several validation steps are incorporated in our work flow.

Antibody selection: Based on sequence homology, only antibodies raised against PrESTs with >60% homology with corresponding mouse genes are selected.

Translational validation: Antibodies exposed to mouse brain lysates using western blot to identify possible off-target interactions with mouse proteins.

Internal comparative validation: If available multiple antibodies raised against different fragments of targeted proteins are applied to mouse brain tissue. Reliability score increases when 2 or more antibodies reveal similar staining patterns.

External multidisciplinary validation: Staining patterns will be evaluated using peer-reviewed published data on cellular and regional distribution of proteins. In addition protein distribution data is assessed using expression data available in the Allen Brain Atlas.

Protein array (PA)

All purified antibodies are analyzed on antigen microarrays. The specificity profile for each antibody is determined based on the interaction with 384 different antigens including its own target. The antigens present on the arrays are consecutively exchanged in order to correspond to the next set of 384 purified antibodies. Each microarray is divided into 21 replicated subarrays, enabling the analysis of 21 antibodies simultaneously. The antibodies are detected through a fluorescently labeled secondary antibody and a dual color system is used in order to verify the presence of the spotted proteins. A specificity profile plot is generated for each antibody, where the signal from the binding to its own antigen is compared to the eventual off target interactions to all the other antigens. The vast majority (86%) of antibodies are given a pass and the remaining are failed either due to low signal or low specificity.

Standard antibody validation - PA

Supported

• Pass with single peak corresponding to interaction only with its own antigen.

Approved

• Pass with quality comment low specificity (binding to 1-2 PrESTs >15% and <40% of the signal from the target PrEST).

Uncertain

• No or weak signal.
• Low specificity (one antigen with >40% signal or more than two antigens with signal >15% of the signal from the target PrEST).

Western blot (WB)

Western blot analysis of antibody specificity has been done using a routine sample setup composed of IgG/HSA-depleted human plasma and protein lysates from a limited number of human tissues and cell lines. A selection of antibodies with an uncertain routine WB have been revalidated using an over-expression lysate (VERIFY Tagged Antigen(TM), OriGene Technologies, Rockville, MD) as a positive control. Antibody binding was visualized by chemiluminescence detection in a CCD-camera system using a peroxidase (HRP) labeled secondary antibody.

Antibodies included in the Human Protein Atlas have been analyzed without further efforts to optimize the procedure and therefore it cannot be excluded that certain observed binding properties are due to technical rather than biological reasons and that further optimization could result in a different outcome.

Standard antibody validation - WB

Supported

• Bands corresponding to the predicted size in kDa (+/-20%).
• Band of predicted size in kDa (+/-20%) with additional bands present.

Uncertain

• Single band larger than predicted size in kDa (+20%) but partly supported by predicted transmembrane region, signal peptide or by other available data.
• No bands detected.
• Single band differing more than +/-20% from predicted size in kDa and not supported by predicted transmembrane region, signal peptide or by other available data.
• Weak band of predicted size in kDa (+/-20%) but with additional bands of higher intensity also present.
• Only bands not corresponding to the predicted size.
• Target too small/large to be analyzed with the present setup.
• Current setup is not applicable due to low RNA count

For antibodies showing uncertain Western blot data the corresponding image is not shown.

Enhanced antibody validation - WB

Genetic validation - siRNA

This method is based on the knock-down or knock-out in a suitable cell line of the target protein using genetic methods, such as CRISPR or siRNA. The staining of the antibody is evaluated by Western blot through analyses of samples from cell lysates before and after knock-down of the corresponding target gene. The results show no or weaker band in the lysate from the knock-down cell line.

Antibodies that meet one of the following criteria will receive the validation score “Enhanced” by genetic method:

• Signal downregulation > 25 % by both siRNAs.
• Signal downregulation > 25 % by one siRNA.

Recombinant expression validation

This method is based on over-expression of the target protein in a cell line preferably not expressing the target protein. The staining of the antibody is evaluated by Western blot through analyses of samples from cell lysates with and without recombinant expression of the target protein. The results show no or weak band from the unmodified cell line lysate and a strong band in the cell line with recombinant expression.

Independent antibody validation

This method is based on comparing the staining pattern using two independent antibodies with no overlapping epitopes. The staining of the two antibodies is compared by Western blot through analyses of samples from at least two cell lysates preferably expressing the target protein at different levels. The results show similar Western Blot patterns achieved with independent antibodies.

Orthogonal validation

This method is based on manual evaluation by comparing the antibody band intensity against the corresponding protein levels quantified by mass spectrometry (MS). Antibodies are considered enhanced where the staining intensity and protein expression levels show the same pattern. At least two cell or tissue samples must be used and the target protein must express the target at different levels. This method can also be used to compare the protein expression levels determined by the antibody with the corresponding RNA in each corresponding cell line or tissue.

Capture MS validation

This method is based on comparison between the molecular weight of the stained band visualized by the antibody against the protein size obtained by a capture MS method in which multiple gel slices are cut out from the electrophoretic separation and analysed separately by proteomics. The proteins in each gel slice are digested into peptides and the protein presence and its migration in the gel is verified after the subsequent proteomics analysis. The band detected by the antibody should be equivalent to the same of the intended target protein and its peptide(s).