Bosterbio's Monoclonal antibodies validation Information

Antibodies are among the most frequently used tools in basic science research and in clinical assays. Currently, there is no comprehensive scientific framework for antibody validation within the research community. As a result, the quality and data reproducibility through the use of these commercially available antibodies vary greatly. The International Working Group for Antibody Validation (IWGAV) has been working hard to optimize performances of antibodies and propose guidelines for antibody validation.

Bosterbio is an antibody manufacturer specializing in high-affinity and high-specificity antibodies. Our antibodies are thoroughly and transparently validated for numerous applications including IHC, WB, ELISA and Flow Cytometry. We use multiple tissues/cell lines, specific controls, and other various methods to verify our antibodies.

At Bosterbio, we take great pride in the art of antibody production. With over 25 years of experience in developing antibodies, we have established a comprehensive antibody development and validation system. Our technical resources are free to access on our website and we share what we learn to the public. This article lists examples of how we development and validate our monoclonal antibodies. Our highly specific monoclonal antibodies have low non-specific cross-reactivity and minimal lot-to-lot variations.

Initial screen tests

ELISA tests are performed repeatedly in various monoclonal development phases. A titration ELISA is performed with each test-bleed after animal immunization. Spleen cells are harvested from the best-responding animals for hybridoma fusion. An ELISA assay is used to screen the positive supernatants. We perform multiple rounds of limiting-dilution subcloning until all wells are growing at the same rate, secreting antibody at the same concentration, and have the same confirmed isotype.

Selected monoclonal antibodies are subjected to multiple further application testing and optimization including but not limited to WB, IHC, and Flow Cytometry.

Specificity tests

Western Blot Specificity Test

The key to proving specificity is often the correct use of controls. Our lab technicians design multiple controls to test specificity of the antibodies. For Figure 1, rat and mouse heart tissues that are known to express FABP3 are chosen as positive controls while liver, spleen and kidney tissues known not to express FABP3 are chosen as negative controls. A single band at the known molecular weight for the target in the positive controls indicates that the antibody is specific to the target protein.


Figure 1: Detection of mouse anti-FABP3 monoclonal antibody

Western blot analysis of FABP3 using anti-FABP3 antibody (M01734). Electrophoresis was performed on a 8% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 50ug of sample under reducing conditions.
Lane 1: rat heart tissue lysates (positive control, high expression)
Lane 2: rat liver tissue lysates (negative control, low or none expression)
Lane 3: rat spleen tissue lysates (negative control, low or none expression)
Lane 4: rat kidney tissue lysates (negative control, low or none expression)
Lane 5: mouse heart tissue lysates (positive control, high expression)
Lane 6: mouse liver tissue lysates (negative control, low or none expression)
Lane 7: mouse spleen tissue lysates (negative control, low or none expression)
Lane 8: mouse kidney tissue lysates (negative control, low or none expression)

After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA for 50 minutes. The membrane was blocked with 5% Non-fat Milk/ TBS for 1.5 hour at RT. The membrane was incubated with mouse anti-FABP3 antigen affinity purified polyclonal antibody (M01734) at 0.2ug/mL overnight, then washed with TBS-0.1% Tween 3 times with 5 minutes each and probed with a goat anti-mouse IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A band was detected for FABP3 at approximately 15KD. The expected band size for FABP3 is at 15KD.

WB is an appropriate initial validation step to determine an antibody’s specificity. However, for monoclonal antibodies generated against a purified protein, they work well for proteins in their native confirmation, but not when they are denatured. Therefore, WB results cannot be used as absolute evidence for these antibodies or for antibodies that recognize folded epitopes. IHC, Flow cytometry, or other assays will also be performed to validate the specificity of antibodies.


IHC Specificity Tests

A negative control, including no primary antibody in IHC is valuable for IHC experiments (Figure 2 & Figure 3). This is to determine if the secondary antibody is binding nonspecifically to the sample tissue, resulting in false positives or nonspecific binding.

Figure 2. IHC analysis of Cytokeratin 19 using anti-Cytokeratin 19 antibody (M02101-2)

Cytokeratin 19 was detected in paraffin-embedded section of human esophagus squama cancer tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml mouse anti-Cytokeratin 19 Antibody (M02101-2) overnight at 4°C. Biotinylated goat anti- mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1021) with DAB as the chromogen.


Figure 3. IHC analysis of Cytokeratin 19 using anti- Cytokeratin 19 antibody (M02101-2).

Cytokeratin 19 was detected in paraffin-embedded section of human esophagus squama cancer tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with PBS buffer overnight at 4°C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1021) with DAB as the chromogen.


Flow Cytometry Specificity Test

For flow cytometry, we include unstained controls and isotype controls in our testing (Figure 4).

Unstained controls

An unstained sample has not been treated with or exposed to any fluorescent reagents. Unstained cells are used to assess cell auto-fluorescence. and set your voltages and negative gates appropriately.

Isotype controls

Isotype controls are designed to assess the level of background staining for flow cytometric cell analysis. An isotype control uses an antibody of the same isotype as the primary antibody, but is specific for an antigen absent from the cells under study. Isotype controls should be used to determine the background due to nonspecific antibody binding.

Figure 4. Flow Cytometry analysis of k562 cells using anti- CRM1 antibody (M01180).

Overlay histogram showing k562 cells stained with M01180 (Blue line).The cells were blocked with 10% normal goat serum. And then incubated with mouse anti-CRM1 Antibody (M01180,1μg/1x106 cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-mouse IgG (BA1127, 5-10μg/1x106 cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was mouse IgG (1μg/1x106) used under the same conditions. Unstained sample (Red line) was also used as a control.

Reproducibility Tests

Our lab technicians regularly compare the new monoclonal antibodies to previously validated standards stated in the literature prior to release. We run reproducibility tests by using the same antibody with different lots on different days. Lot-to-lot consistency is demonstrated by the combination of test result images of two or more pre-qualified individual lots (Figure 5). We openly describe our procedures and the exact materials used to ensure that customers can get the same great results we did.

Figure 5: Lot-to-Lot reproducibility of mouse anti-TCP1 monoclonal antibody

Western blot analysis of TCP1 using anti-TCP1 antibody (M02389) Electrophoresis was performed on a 8% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 50ug of sample under reducing conditions. Lanes: Human A549 and PANC-1 whole Cell lysates, After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA for 70 minutes. Blocked the membrane with 5% Non-fat Milk/ TBS for 1.5 hour at RT. The membrane was incubated with mouse anti- TCP1 antigen affinity purified polyclonal antibody (M02389) at 0.2ug/mL overnight, then washed with TBS-0.1% Tween 3 times with 5 minutes each and probed with a goat anti-mouse IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system.A band was detected for TCP1 at approximately 60KD.The expected band size for TCP1 is at 60KD.

Summary

Bosterbio is committed to select the best clones and perform extensive in-house validation tests for our monoclonal antibodies. It provides a high level of confidence that result images and data obtained will be highly specific to the target proteins and will be reproducible among various lots.