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Evaluation of LGR5 protein expression in colon cancer stem cells AACR 2016 Abstract
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Home Gene of the month ERCC1|
Validation of a Highly Specific ERCC1 Antibody with Protein Microarray Technology
High specificity is the pre-requisite for any antibody to be used for diagnostic and therapeutic applications. Antibody cross-reactivity will create unexpected side effects or false diagnostic reports for clinicians. Research data from various groups has shown that some monoclonal antibodies on the market are not mono-specific. Similar epitopes are sometimes found across multiple un-related proteins.1
Protein microarray technology is a great platform to evaluate antibody specificity at the proteome-wide level. With the world’s largest collection of overexpression antigen standards, OriGene developed a high density protein microarray for antibody validation. This protein chip is spotted with 10,464 unique overexpression proteins in duplicates on a single nitrocellulose coated glass slide (Figure 1). OriGene’s protein microarray technology has been used to validate the specificity of an existing ERCC1 diagnostic monoclonal antibody, and has been applied as a screening method to identify the most specific TrueMAB™ monoclonal antibody for this target.
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 chemotherapy2. 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 lines3. By using OriGene’s protein microarray technology, the corresponding cross-reactive protein for the 8F1 monoclonal antibody was identified (Figure 2). Most importantly, this technology enabled OriGene to successfully develop a highly specific TrueMAB™ monoclonal antibody for ERCC1 (TA500634 (4F9) (Figure 3). This data was further confirmed by western blot analysis (Figure 4).
Figure 1. Over-expression Protein Lysate Microarray
OriGene’s overexpression lysate protein microarray chip comprises of over 22,000 spots. It includes 10,464 unique protein lysates printed in duplicates and large selections of positive and negative controls.
The array was manufactured as indicated and tested with 1:500 dilution of OriGene anti-DDK (TA50011) tag antibody followed by DyLight 649 conjugated goat anti-mouse IgG secondary antibody.
Figure 2. The identification of 8F1 cross-reactive protein with protein microarray chip.
The OriGene overexpression protein microarray chip was immunostained with the most commonly used 8F1 monoclonal anti-ERCC1 antibody. The positive reactive proteins are pointed with red arrows. This data shows that 8F1 recognizes not only its specific target (two ERCC1 transcript variants), but also another unrelated cytosolic protein 8F1. (The identity of this protein will be revealed in our future publication.)
Figure 3. To screen for the most highly specific anti-ERCC1 monoclonal antibody with protein microarray chip.
The overexpression protein microarray chip was immunostained with TA500634 OriGene TrueMAB monoclonal anti-ERCC1 antibody (4F9). This data shows that 4F9 is highly specific with ERCC1 (pointed with red arrows). No cross-reactivity was observed with any other protein.
Figure 4. Western blot validation of protein microarray chip data.
Eight OriGene VERIFYTM overexpression lysate antigen standards (Lane 1 to 8) and an empty vector transfected HEK293T cell lysate (Lane 9) were fractionated on SDS-PAGE, and then immunoblotted with 8F1 (Upper panel) or 4F9 (Middle panel) anti-ERCC1 monoclonal antibodies separately. The recombinant protein expression level within the lysates were analyzed with anti-DDK antibody (Lower panel). Lanes 1 to 8 are samples for ERCC1 (NM_202001), ERCC1 (NM_001983), ERCC2 (NM_000400), ERCC8 (NM_001007234), ERCC3 (NM_000122), ERCC4 (NM_005236), ERCC5 (NM_000123) and protein 8F1. Lane 9 was loaded with an empty vector transfected HEK293T cell lysate.
- Michaud, G. A., Salcius, M., Zhou, F., Bangham, R., Bonin, J., Guo, H., Snyder, M., Predki, P. F., and Schweitzer, B. I. (2003) Nat Biotechnol 21(12), 1509-1512
- Olaussen, K. A., Dunant, A., Fouret, P., Brambilla, E., Andre, F., Haddad, V., Taranchon, E., Filipits, M., Pirker, R., Popper, H. H., Stahel, R., Sabatier, L., Pignon, J. P., Tursz, T., Le Chevalier, T., and Soria, J. C. (2006) N Engl J Med 355(10), 983-991
- Bhagwat, N. R., Roginskaya, V. Y., Acquafondata, M. B., Dhir, R., Wood, R. D., and Niedernhofer, L. J. (2009) Cancer Res 69(17), 6831-6838
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