Advanced Validated Antibody     Validated Antibody Methods

ProSci uses a 2 part approach for advanced validation antibodies.

Part 1- Verifying target specificity: In order to ensure functionality, at least one of the following antibody validation methods is tested.

Genetic Strategies (GS)

siRNA Knockdown expression testing using RNAi to knockdown gene of interest.In mammalian cells, short pieces of double-stranded RNA known as short interfering RNA (siRNA), initiate the degradation or knockdown of a specific, targeted cellular mRNA. In this process, the antisense strand of the siRNA duplex becomes part of a multi-protein complex called the RNA-induced silencing complex (RISC). RISC then identifies the complementary mRNA and cleaves it at a specific site. Next, this cleaved message is targeted for degradation, ultimately resulting in the loss of protein expression. Here at ProSci to further validate quality of Antibodies, target protein siRNAs are transfected into the cell lines that highly express the target protein. After gathering information on our top priority antibodies, DNA oligos are designed for target genes. siRNAs are synthesized for target genes. siRNA transfection will be performed on highly expressed cell lines. After transfection, the control and KD lysates are tested by western blot using antibodies against different epitopes for the same protein.  If tested antibodies show the KD effect they be added to the list of validated products.

Independent antibody verification (IAV):

Target expression is performed using two differentially raised antibodies recognizing the same protein target to test for antibody specificity. In other words the two antibodies are used that target non-overlapping epitopes of an antigen. By obtaining comparable results from antibodies that recognize independent regions of the same target protein, this allows for increased confidence that these antibodies are specific and suitable for the detection of their intended target. Common applications of independent antibody validation would be obtaining similar detection patterns in multi-lysate western blots, IHC arrays, immunofluorescence of multiple cell lines, immunoprecipitation, flow cytometry, and other antibody applications. Results of IAV can vary depending on sample prep, buffer systems, orientation in a multi-protein complex and other parameters that can influence protein conformation.

Immunoprecipitation-mass spectrometry (IP-MS): 

Immunoprecipitation in combination with mass spectrometry (IP-MS) will verify that an antibody interacts specifically with an intended target. Once target antibody is chosen for validation by IP-MS analysis, candidate cell lines will be decided based on their capabilities to express the target protein. Cell lysate is prepared and an optimized immunoprecipitation is carried out, and the resulting IP eluate samples are then prepared and analyzed by mass spectrometry. MS data set is processed using bioinformatics analysis software. Analysis  involves: (1) differentiating true positives from negative controls and background, and (2) calculating fold enrichment to evaluate direct (IP) and indirect (co-IP) products by comparing the abundance and enrichment of the target relative to off-target proteins. Only limitation with IP-MS is that not all antibodies will immunoprecipitate proteins from cell lysates. An antibody may not recognize the native form of its target protein. This does not make the antibody a bad choice for use in other applications. However, it does make it impossible to verify the antibody specificity in IP-MS. Other methods are required to verify these antibodies, including genetic modification (knockout or knockdown), biological activation, and other methods. Analysis of IP-MS antibody validation data is carried out with two different antibodies: Target antibody, and at least one well-characterized control antibody that is specific to an unrelated target. The MS signal intensity of each protein identified by MS is then plotted with respect to       the two IP antibodies. The proteins fall into three distinct groups

  • Background—proteins that are present in both samples are found in the central diagonal region of the graph. These are background proteins that bind either to antibodies in general or to the resin used in the immunoprecipitation.
  • Negative control—proteins that only bind to the negative control antibody are found along the x-axis (i.e., y-value is essentially zero). These are negative control proteins, which do not interact with the antibody of interest.
  • Positive—proteins that bind only to the antibody of interest are found along the y-axis (i.e., x-value is essentially zero). These are positive proteins, which are specifically immunoprecipitated by the antibody of interest.
Orthogonal strategies

Correlation using both antibody dependent and independent method. In this enhanced validation method, the antibody is validated by comparing the results with a non-antibody based method across multiple samples. One orthogonal approach is to compare antibody staining intensities to RNA-Seq data from the same samples, using multiple tissues or cells with varriable expression of the target protein. Antibody specificity is confirmed when the antibody signal matches RNA levels in the evaluated samples. For each antibody, two tissues or endogenous cell lines are chosen for the validation, one with high RNA expression and the other with low or no RNA expression of the target. Enhanced validation by comparing the antibody signal to RNA-Seq data is used to validate antibodies in WB and IHC. The method can also be applied for ICC-IF.Orthogonal Validation by Comparing Antibody Signal in Western Blot to RNA: In Orthogonal validation by comparing antibody signal in Western blot to RNA, the Western blot result will be compared with RNA-Seq data for the same samples, using both positive and negative samples.Antibody specificity is confirmed when the antibody signal matches RNA levels in the evaluated samples. For each antibody, two tissues or endogenous cell lines are chosen for the validation, one with high RNA expression and the other with low or no RNA expression of the target. The cell lines are chosen so that there is a significant difference between the RNA expression in the high and low samples. In the enhanced validation data presented for the antibodies, the Western blot lanes in the high and low cell lines are displayed together with their corresponding RNA values. 

Recombinant Expression(RE)

In this enhanced validation method, the antibody binding is verified using and over-expressed or tagged version of the target protein. When over-expressing the target protein in a cell line, the antibody is validated by comparing the signal from the over-expressed version with the unmodified endogenous target protein. This approach can be applied in WB. The method is applied to Western blot by comparing the antibody signal in a sample where the target protein has been recombinantly over-expressed, to a control sample. Antibody specificity is confirmed when the antibody shows a strong band in the cell line with recombinant expression and no or faint band in the control.

In the validation data presented for the antibody, the Western blot includes the over-expressed sample and the control sample in the same blot.  

Cell treatment—detecting downstream events following cell treatment
Relative expression—using naturally occurring variable expression to confirm specificity.
Neutralization—functional blocking of protein activity by antibody binding
Peptide array—using arrays to test reactivity against known protein modifications

Part 2- Functional application validation: ensuring the antibody works in a particular applications using following techniques:

  1. ELISA
  2. Western blot
  3. Flow Cytometry
  4. immunohistochemistry /immunocytochemistry (IHC/ICC)
  5. immunofluorescence (IF)​

Selecting a detector and capture antibody that are target specific is very crucial. Only pairs with high specificity, selectivity, and consistent linearity of dilution for the target will be used for further development. Plasma, Serum, or tissue lysate will be used to validate antibody selectivity. In this method purified recombinant target proteins will be added into the biological matrix, which should be recovered and detected. The recovery observed for the spike should be almost identical in both the biological matrix and the standard diluent for a sample matrix to be considered valid for our ELISA assay.

  • Western blot

Lysates from cells or tissues that have been identified to express the protein of interest will be used to validate antibodies in western blot by comparing the expected molecular weight. Positive and Negative controls will be always run in the same western blot experiment. We will always run several controls in the same western blot experiment, including positive lysate and negative lysate. When possible, we also include knock-out (KO) cell lines as a true negative control for our western blots. This isn’t possible for all the antibodies on our catalog; however, we are always increasing the number of KO-validated antibodies we provide. In addition, we run old stock alongside our new stock. If we know the old stock works well, this also acts as a suitable positive control. If the western blot result gives a clear clean band and we are happy with the result from the control lanes, these antibodies will be passed and added to the catalog. 

  • Flow Cytometry

When validating antibodies for flow cytometry, we optimize staining protocol, including fixation, permeabilization, and washing. We include relevant controls routinely in our experiment to determine non-specific binding of an antibody.  

 Flow cytometry analysis of monoclonal antibodies is performed to identify if a monoclonal antibody has binding activity to the target protein expressed in transfected cells, and also determine the optimal binding concentration of the antibody.  An antibody titration analysis is performed during the initial characterization of antibody to determine at what concentration the antibody shows optimal binding.Optimal binding concentration is defined as the lowest concentration where the antibody binds in a saturating manner as indicated by a cell population shift depicted in a flow cytometry histogram overlay.  This shift is measured by comparing antibody binding between transfected and untransfected HEK cells.

The optimal binding concentration may be different for different antibodies as is determined at the antibody’s initial release. Histogram overlay records are also retained on the flow cytometer for comparison. If a new lot of antibody shows the same effective concentration within two fold (i.e. one dilution), the antibody will be released. If after a second test the antibody does not pass, a new lot of antibody will be purified and tested.

  • Immunohistochemistry (IHC/ICC)

IHC and ICC are fantastic for validating whether an antibody recognizes the correct protein, based on cellular and subcellular localization. Antibody specificity is confirmed by looking at cells that either do or do not express the target protein within the same tissue.  We validate antibodies in IHC by using first or second bleed affinity purified using either sulfhydryl-ligand immobilized affinity column or amine-ligand immobilized affinity column. Yolk is used for cases where the product is a chicken IgY antibody. In order to confirm presence of antibodies we perform ELISA on non-purified bleeds, purified antibody, and flow-throughs to confirm presence of antibody.

 If protein bands are the expected size and in correct tissues then appropriate tissue or cell slides are selected for Immunohistochemistry or Immunocytochemistry) analysis based on the western blot result or from literature. Primary antibody sensitivity will be tested with the different dilution in BSA diluents. The best IHC/IF images will be scanned under the microscope. The new antibody can be released if meets ProSci QC criteria.

  • Immunofluorescence (IF):

New Polyclonal antibodies are validated by IF.  Immunofluorescence (IF) microscopy is generally used by researchers to assess both the localization and endogenous expression levels of proteins of interest. Immunofluorescence can be applied on tissue sections, cultured cells or individual cells that are fixed by a variety of methods. Antibodies can be used in this method to analyze the distribution of proteins, glycoproteins and other antigen targets including small biological and non-biological molecules. The effective application of this method comprises several considerations, including the nature of the antigen, specificity and sensitivity of the primary antibody, properties of the fluorescent label, permeabilization and fixation technique of the sample, and fluorescence imaging of the cell. Although each protocol will require fine‐tuning depending on the cell type, the antibody, and the antigen, there are steps common to nearly all applications.