Antibody validation

The usefulness of antibodies in different assays is dependent on both sensitivity and specificity of epitope binding. In order to assure the quality of the antibodies used in the Human Protein Atlas, they are validated by a set of defined criteria. All assays and annotations used in the Human Protein Atlas are described below.

Standard antibody validation

All antibodies produced within the Human Protein Atlas project (HPA-antibodies) must pass steps 1-3 in the list below in order to be used for immunohistochemistry and Immunocytochemistry/IF. Steps 4-6 provide a basis for an evaluation and scoring of antibody validity. 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:

Quality assurance steps for antibodies generated within the Human Protein Atlas project:

1. Plasmid inserts are sequenced to assure that the correct protein epitope signature tag (PrEST) sequence is cloned.
2. Size of resulting recombinant protein (including the specific PrEST) is analyzed using mass spectrometry to assure that the correct antigen has been produced and purified.
3. 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.
4. 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 a uncertain routine WB have been revalidated using an over-expression lysate as a positive control.
5. Immunohistochemical staining of normal and cancer tissue is examined by trained personnel to assure plausible immunohistochemical staining properties.
6. High resolution confocal microscopy images of immunofluorescently stained human cell lines are annotated for specific subcellular localizations by trained cell biologists, and the subcellular localization patterns are compared with the immunohistochemical staining and available experimental gene/protein characterization data.

For commercially available antibodies (CABs), immunohistochemistry has been performed in a similar manner as for HPA-antibodies. These antibodies have also been tested on Western blots. For each commercially available antibody, a link to the antibody provider is given on the "Antibody validation" page.

For antibodies supplied through commercial or other academic sources we provide Western blot validation, immunofluorescence validation and immunohistochemistry validation based on literature conformity and for immunohistochemistry validation also RNA consistency. For further validation we refer to quality controls provided by the respective company.

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

Formally validated antibodies

Antibodies used in the Human Protein Atlas are formally validated based on the pillars presented in "A proposal for validation of antibodies" (Uhlen et al 2016). Antibodies that fullfil the criteria are labelled "Validated". The following Formal antibody validations are used for each assay:

• Immunocytochemistry: Genetic method, Tagged proteins, and Independent antibodies
• Immunohistochemistry: Orthogonal method and Independent antibodies
• Western blot: Genetic method

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

	Genetic method: the antibody specificity has been validated by siRNA knockdown of the target protein prior to Immunocytochemistry/IF or Western blot analysis.
	Orthogonal method: the antibody specificity has been validated by comparing an antibody-based method with an antibody-independent method; in this case protein levels are compared to RNA levels.
	Independent antibodies: the antibody specificity has been validated by comparing two or more antibodies that recognize different epitopes on the target protein.
	Tagged proteins: the antibody specificity has been validated by co-staining of a cell line expressing the target protein tagged with GFP at near-endogenous levels.

Immunocytochemistry/IF - cells

Standard antibody validation - ICC/IF

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.

Formally validated antibodies - ICC/IF

Validation score for Immunocytochemistry/IF - formal validation:

Validated

• The observed location(s) is(are) supported by an additional validation method; Genetic method (siRNA knockdown), Tagged protein (fluorescent tagged protein), and Independent antibodies.

Genetic method - siRNA

To validate the protein subcellular localization determined with the antibody, the staining procedure is repeated on siRNA transfected U-2 OS cells to knock-down the expression of the protein Stadler 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 fixated 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 down regulation. The distribution of RFI for the cells in 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 validation assay a validation score is assigned based on the decrease in antibody-based staining intensity upon target protein downregulation.

Validation scores for Immunocytochemistry/IF siRNA validation:

Validated

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

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, Dresden, (Poser et al 2008; Skogs et al 2016). They are produced using a Bacterial Artificial Chromosomes (BAC) TransgeneOmics technology where transfection with BAC allows for a large transgenic insert with all regulatory elements present resulting in near-endogenous expression. 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 detect even low abundant tagged target protein.

All images are manually annotated to one or several subcellular locations and if applicable combined with parameters describing the staining characteristics (e.g. smooth, granular, speckled or fibrous).

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.

The location of the tagged protein is taken into account when performing the knowledge-based annotation of subcellular location for each gene described here.

For each GFP validation assay a validation score is assigned based on colocalization of the antibody staining and the GFP-tagged protein.

Validation scores for Immunocytochemistry/IF GFP validation:

Validated

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

Independent antibodies

The protein localization(s) can be validated using independent antibody strategies. Two (or more) independent antibodies directed towards different 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" antibody(ies).

Validation scores for Immunocytochemistry/IF Independent antibodies

Validated

• Two independent antibodies yielding the same staining pattern for main and additional localization.

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 the expression pattern 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 a protein level and that a substantial quantity of published experimental data is available from literature and public databases. Limited protein/gene characterization data does not require evidence of existence on a 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 with internally and externally generated RNA-seq data.

The different levels of validation score are Supported, Approved or Uncertain.

Validation scores for Immunohistochemistry:

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

Formally validated antibodies - IHC

Orthogonal method

Cell lines
For all single-target antibodies on genes with supportive tissue atlas data, the Pearson correlation between estimated RNA expression and protein expression across a set of 44 cell lines is calculated. Continuous values of protein expression based on TMAx and TPM values obtained from RNA-seq are correlated, with a Pearson's r >0.6 resulting in a formal validation for the antibody for use in immunohistochemistry.

Tissue
For all single-target antibodies on genes with supportive tissue atlas data, the Spearman correlation between estimated RNA expression and protein expression across a set of 37 tissues is calculated. Protein expression values based on antibody staining and TPM values obtained from RNA-seq are correlated, with a Spearman's rho >0.6 resulting in a formal validation for the antibody for use in immunohistochemistry. All TPM values less than the detection limit of 1 were set to 0 for the correlation calculation.

Independent antibodies

Cell lines
For genes with supportive tissue atlas data where at least two in-house generated single-target antibodies are available (to ensure binding to different epitopes) the Pearson correlation comparing protein expression across a set of 46 cell lines for each antibody pair is calculated. Continuous values of protein expression based on TMAx for the antibodies are correlated, with a Pearson's r >0.6 resulting in a formal validation for the antibody for use in immunohistochemistry.

Tissue
For genes with supportive tissue atlas data where at least two in-house generated single-target antibodies are available(to ensure binding to different epitopes), the Spearman correlation comparing protein expression across a set of 76 cell types for each antibody pair is calculated. Protein expression based on antibody staining for the antibodies are correlated, with a Spearman's rho >0.6 resulting in a formal validation for the antibody for use in immunohistochemistry.

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.

Formally validated antibodies - WB

Genetic method - siRNA

Western blot analysis for each antibody is scored as validated if the signal from one of or both siRNA lane/s are >25% weaker than from the control lane. Total protein amount in all lanes are taken in consideration when scoring.

Validated

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