UltraMAB®, the Ultra Specific Antibody
Validated against > 10,000 human antigens
Performance and specificity are the pre-requisites for antibodies to be used for diagnostic and therapeutic applications. To ensure the superior performance, every UltraMAB® monoclonal antibody is validated according to the scientific findings and the medical records of related diseases. Major applications of validation include WB, IHC staining with over 25 types of normal and cancer human tissues, IF/ICC, and FACS.
Some commonly used diagnostic antibodies perform well in applications but cross-react with other unrelated proteins. This cross-reactivity may potentially cause unexpected side effects and generate false diagnostic reports for clinicians. However, the tools to test the mono-specificity are lacking and remain a challenge to the antibody market.
A high density protein microarray chip has been developed for antibody specificity testing. Using the chip spotted with 11,088 (10K) unique over-expressed proteins, we have validated the specificity of existing HER2 and ERCC1 diagnostic antibody (View publication on the validation of ERCC1 antibodies
). This protein microarray technology has also been applied to identify UltraMAB™, the most specific antibodies for cancer biomarkers and other important diagnostic targets.
UltraMAB Currently Available: (New! 30ul trial size - please request)
UltraMAB specificity validation: 10K high density protein microarray
Figure 1. Diagram of a 10K high density protein microarray
Protein microarray slide specifications:
||White nitrocellulose glass slide
|Slide NC pad dimension:
||20mm x 60mm
||48 (4 columns x 12 rows)
||4200um x 4400um
||21 rows (Vertical) x 22 columns (Horizontal)
|Median Spot Diameter:
|Spot Center to Center Spacing:
|Distances Between Subarrays:
|Replicates per Sample:
Under current specifications, 11,088 samples were spotted in duplicates on a single nitrocellulose slide. These include 10,380 unique overexpression lysates and 708 positive and negative control samples. Serial diluted mouse, human and rabbit IgG mixture as positive controls were spotted on three subarrays highlighted in redboxes. Subarrays highlighted in yellowboxes contain serial diluted HEK293T cell lysates as negative controls. All the subarrays contain standard positive and negative controls as shown in the figure.
Case Study 1: HER2 (also called ERBB2) Pathology Antibodies
Figure 2. Specificity test on 10K protein chips with antibody 4B5 (left) and HER2 UltraMAB UM500036 (right)
"A semi-quantitative immunohistochemical assay using anti-HER2 antibody is applied to determine HER2 protein overexpression in breast cancer tissues. The specificity of the HER2 antibody (e.g. Clone4B5) is critical because the test results will help oncologist decide a patient should receive Herceptin® treatment. By using the 10K high-density protein microarray, we have revealed that the antibody 4B5 is not specific to HER2 protein. As shown in the graph above, this antibody also reacts with ZSCAN1B and HER4 (ERBB4). In contrast, HER2 UltraMAB (Clone UMAB36) only recognizes HER2 protein and is thus specific"
Case Study 2: ERCC1 IHC antibodies
Figure 3. Specificity test on 10K protein chips with 8F1 and ERCC1 UltraMAB (4F9)
"The excision repair cross-complementation group 1 (ERCC1) protein is an important biomarker for clinicians to predict whether certain patient populations with non-small cell lung carcinoma will respond to cisplatin chemotherapy. As such, it is critical to develop highly-specific immunohistochemistry validated monoclonal antibodies for this diagnostic test. Several publications revealed that 8F1, the most commonly used antibody clone for ERCC1, exhibits cross-reactivity to an unknown protein in ERCC1 deficient cell lines. By using the high-density protein microarray technology, the corresponding cross-reactive binding protein for 8F1 monoclonal antibody was identified. This technology also enable us successfully develop the most specific UltraMAB™ monoclonal antibody for ERCC1 (4F9, Clone UMAB8). This data was further confirmed by western blot analysis. Review the publication on the validation of ERCC1 antibodies."
Figure 4. Western blot validation of protein microarray chip data.
Figure 5. IHC staining of ERCC1 UltraMAB (clone 4F9) of a pathologically validated NSCLC FFPE tissue sections.
Disclaimer: UltraMAB® is a registered trademark of Origene Technologies Inc.