Neutralization, or functional blocking, of proteins by antibodies is often used to study biologic function mediated by cell surface expressed proteins. For example, to study the effects of a particular cytokine on cell signaling and other biologic effects, antibodies can be used that will specifically bind and reduce the amount of free cytokine that can bind to receptors; thereby, down regulating its biologic effects. Neutralization testing is one strategy we use to validate Invitrogen antibodies for research use. When used as a tool to test the specificity of antibodies, the rationale is that when an antibody exhibits functional blocking characteristics it is strong evidence that the antibody is binding the intended target and demonstrating that the antibody is specific to its intended target. To determine if neutralization or a functional blocking approach is applicable for specificity testing, one has to rely on a defined system with a measurable output in order to be able to quantify the effects of the blocking function. These systems could be a motility assay, bioactivity assay, or any other measurable activity.

Advanced Verification

The Advanced Verification badge is applied to products that have passed application and specificity testing. Advanced Verification testing of Invitrogen antibodies continues to expand across the portfolio. An antibody lacking the badge should be seen as an antibody that has not yet undergone testing – not a reflection of the specificity of the product. This badge can be found in the search results and at the top of the product specific webpages. Data supporting the Advanced Verification badges can be found in product specific data galleries.

Neutralization/functional blocking validation data

In the example below (figure1 A), recombinant Red firefly luciferase generates light (bioluminescence) in the presence of luciferase substrate (left panel). The Red firefly luciferase antibody blocks RFF luciferase activity in a dose-dependent manner, resulting in decreased activity as reflected by reduced bioluminescence.

When possible, we combine blocking testing with other supporting data, like a western blot, to show that the antibody recognizes the intended protein target at the expected molecular weight (right panel). In the example (figure 1 B), whole cell lysates from Red firefly (RFF) luciferase, Green Renilla (GR) luciferase, or untransfected 293 cells were analyzed in a western blot. As expected, the Red firefly luciferase antibody only detected its target in cells expressing the Red firefly luciferase protein. From these results we can verify that the Red firefly luciferase antibody functions as expected.

Neutralizing effect of Red firefly

Figure1 A. Neutralizing effect of Red firefly (RFF) luciferase antibody on the activity of purified recombinant RFF luciferase (from the Japanese firefly Luciola cruciata) using the Thermo Scientific Firefly Luc One-Step Glow Assay. The recombinant enzyme (10 ng) was incubated with the indicated amounts of a RFF luciferase polyclonal antibody (Cat. No. PA1-179) for at least 30 min. Enzyme activity (RLU) was detected using a Thermo Scientific Varioskan Flash Reader and the Firefly Luc One-Step Glow Assay Kit (Cat. No. 16197). Average RLU values (± standard deviation) from 10 time points per concentration of 4 replicates is shown.

Western blot analysis of Red firefly luciferase

Figure 1 B. Western blot analysis of Red firefly luciferase (from the Japanese firefly Luciola cruciata). Electrophoresis was performed after loading 10 µg of whole cell lysates from untransfected (UT) and Red firefly (RFF Luc) or Green Renilla (GR Luc) luciferase transfected 293 cell lines, and 10 µL of PageRuler Plus Prestained Protein Ladder (Cat. No. 26619) onto a 4–20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked with 5% BSA/TBST for 1 hour. The membrane was probed with a RFF luciferase polyclonal antibody (Cat. No. PA1-179) at a dilution of 1:4,000 overnight at 4°C on a rocking platform, washed in TBS-0.1% Tween-20, and probed with a prediluted goat anti–rabbit IgG HRP secondary antibody (Cat. No. 32460) at a dilution of 1:500 (20 ng/mL) for 1 hour. Membranes were washed, and chemiluminescent detection was performed using SuperSignal West Pico substrate (Cat. No. 34080).

In addition to the neutralization of biological activity for specificity testing of antibodies, blocking can be utilized in a different way to determine specificity. In these cases, the immunizing peptide, which is usually abundantly available, can be used as a method for antibody specificity verification. We routinely use this control in western blotting applications, especially when performing specificity testing of antibodies that are directed against specific phosphorylation sites.

Verifying target specificity of Invitrogen antibodies using neutralization/blocking

Invitrogen antibodies that have been verified against neutralization/blocking are indicated with a “verified specificity” symbol in search results and on relevant product pages. The data showing the verification will be provided on each product page.

*The use or any variation of the word “validation” refers only to research use antibodies that were subject to functional testing to confirm that the antibody can be used with the research techniques indicated. It does not ensure that the product(s) was validated for clinical or diagnostic uses.