Validation controls

As a resource for antibody validation, we strongly encourage users and antibody providers to upload data comprising both negative and positive controls, in addition to an experimental result. Dissemination and transparency allows for objective interpretation of antibody performance and specificity.

Gel shift

1. Demonstrate conditions under which gel shift does not occur despite incubation of oligonucleotide with protein sample (expect to see one or two of a-c)
   1. (radiolabelled) wildtype oligonucleotide probe incubated with antigen-depleted lysate or other protein sample
   2. (radiolabelled) scrambled oligonucleotide probe incubated with wildtype lysate or other protein sample
   3. radiolabelled oligonucleotide probe incubated with wildtype lysate or other protein sample; interaction competed with excess cold probe 
2. Demonstrate conditions under which supershift does not occur despite formation of antigen-oligonucleotide complex (expect one of a, b)
   1. non-immune or pre-immune serum added to antigen-probe complex
   2. nonspecific (isotype-matched) control antibody added to antigen-probe complex
3. Additional controls: alternate conditions under which supershift occurs (for example, independent/ previously validated positive-control antibody added to antigen-probe complex)

Flow cytometry

1. Demonstrate conditions under which incubation with antibody does not evoke increased fluorescence of sample cell population (expect one of a-d)
   1. cells are incubated with isotype-matched negative-control/ non-binding antibody
   2. cells are incubated with antibody of interest and blocking peptide
   3. cells not expressing antigen are incubated with antibody of interest (in the instance that other controls are not available, since not applicable to all antigens)
   4. non-permeabilized cells are incubated with antibody of interest (intracellular antigen)
2. Additional controls: alternate conditions under which fluorescence increases
   1. cells are incubated with independent/ previously validated antibody against same target

Chromatin IP/ RNA IP

1. Demonstrate conditions under which antigen/ nucleic-acid interaction is not detectable (expect b, but a also possible)
   1. immunoprecipitation performed in cell-type depleted for antigen or with non-post-translationally modified antigen
   2. immunoprecipitation performed under conditions nonspecific for antigen immunoprecipitation
      1. non-immune or pre-immune serum
      2. nonspecific (isotype-matched) antibody
2. Verify specificity of antigen/ nucleic-acid interaction (antigen binding to control nucleic-acid sequences; expect to see a and one of b-d)
   1. control PCRs (to assess antigen association with previously documented sequences) performed in samples positive for antigen and subjected to immunoprecipitation with antibody of interest
   2. control PCRs performed in samples depleted for antigen but subjected to immunoprecipitation with same antibody
   3. control PCRs performed in samples expressing antigen but subjected to immunoprecipitation with nonspecific (isotype-matched) antibody or pre-/ non-immune serum
   4. control PCRs with non-immunoprecipitated (input) nucleic acid
3. Additional controls: alternate conditions to verify antigen/ nucleic-acid interaction
   1. control PCRs performed in samples containing antigen and subjected to immunoprecipitation with an independent or previously validated antibody

Immunoprecipitation

1. Demonstrate specificity of antibody of interest's interaction with antigen (expect one or two of a-c)
   1. immunoprecipitation of sample expressing antigen with nonspecific (isotype-matched) antibody
   2. immunoprecipitation of sample expressing antigen with pre-immune/ non-immune serum
   3. immunoprecipitation of sample lacking antigen expression or expressing non-post-translationally modified antigen with antibody of interest
2. Protein standard
3. Additional controls: alternate conditions for antigen immunoprecipitation
   1. confirmation that independent/ previously validated Ab immunoprecipitates the same band(s))

Western blot

1. Provide assessment of nonspecific bands recognized by antibody of interest (expect one of a, b)
   1. blotting of control sample lacking antigen or with non-post-translationally modified antigen
   2. blot probed with pre-/ non-immune serum or nonspecific (isotype-matched) antibody
2. Demonstrate alternate conditions for antigen detection (expect either a or b)
   1. blot probed with independent antibody against antigen
   2. blot probed with antibody of interest; positive-control antigen sample (recombinant antigen or positive-control lysate/ homogenate) run in parallel with experimental samples
3. Protein standard

Microscopy

1. Demonstrate antigen localization (expect one of a, b)
   1. image of staining with antibody of interest and counterstain
   2. image of staining with antibody of interest and costain for organelle-specific marker
2. Optional: assess antibody specificity
   1. image of staining with competing peptide against antibody of interest
   2. image of staining with nonspecific (isotype-matched) antibody/ pre-immune or non-immune serum
   3. image of staining with antibody of interest in cell-type not expressing antigen
3. Additional controls: alternate conditions for antigen detection
   1. independent image demonstrating localization of Ag-FP fusion
   2. image of staining with independent/ previously validated antibody

Blocking/ neutralizing antibodies

1. Demonstrate proof that antibody of interest neutralizes pathogen entry into cell (expect at least one of a-d)
   1. assay with (isotype-matched) antibody nonspecific for antigen
   2. assay with low-affinity antibody against antigen
   3. assay with antibody of interest in antigen-deficient cell type
   4. assay with competing peptide against antibody of interest
2. Antibody dilution series to show diminishing effect of antibody of interest against pathogen entry into cell
3. Additional controls: assay with alternate conditions for antigen detection
   1. independently validated positive-control neutralizing antibody

ELISA

1. Demonstrate specificity of antigen-antibody interaction (expect at least one of a-d)
   1. assay with sample incubated with (isotype-matched) nonspecific antibody
   2. assay with sample incubated with low-affinity antibody
   3. assay with sample depleted for antigen or with non-post-translationally modified antigen
   4. assay with sample incubated with competing peptide against antibody of interest
2. Antigen or antibody standard curve for interpretation and quantitation of results
3. Additional controls: alternate conditions for antigen detection
   1. assay with independent/ previously validated high-affinity or other control antibody

Radioimmunoassay

1. Demonstrate specificity of antigen-antibody interaction (expect at least one of a-d)
   1. assay with sample incubated with (isotype-matched) nonspecific antibody
   2. assay with sample incubated with low-affinity antibody
   3. assay with sample depleted for antigen or with non-post-translationally modified antigen
   4. assay with sample incubated with competing peptide against antibody of interest
2. Standard curve with different concentrations of radiolabelled antigen incubated with antibody for interpretation and quantitation of results
3. Additional controls: alternate conditions for antigen detection
   1. assay with independent or previously validated high-affinity or other control antibody

Protein array

1. Assessment of antibody specificity and identification of nonspecific interactions (expect a, b)
   1. for antibody array, control array incubated with sample depleted for antigen of interest
   2. for lysate/ antigen array, control array incubated with nonspecific (isotype-matched) antibody
2. Additional controls: alternate conditions for antigen detection (expect a, b)
   1. independent/ previously validated antibodies printed on antibody array
   2. positive-control antibodies or serum samples for antigen array