title: Using protein microarray technology to screen anti-ERCC1 monoclonal antibodies for specificity and applications in pathology.
abstract: BACKGROUND
  An antibody with cross-reactivity can create unexpected side effects or false diagnostic reports if used for clinical purposes. ERCC1 is being explored as a predictive diagnostic biomarker for cisplatin-based chemotherapy. High ERCC1 expression is linked to drug resistance on cisplatin-based chemotherapy. 8F1 is one of the most commonly used monoclonal antibodies for evaluating ERCC1 expression levels in lung cancer patient tissues, but it has been noted that this antibody cross-reacts with an unknown protein.
  
  
  RESULTS
  By using a high density protein microarray chip technology, we discovered that 8F1 not only reacts with its authentic target, ERCC1, but also cross-reacts with an unrelated nuclear membrane protein, PCYT1A. The cross-reactivity is due to a common epitope presented on these two unrelated proteins. Similar to the subcellular localization of ERCC1, IHC tests demonstrated that PCYT1A is localized mainly on nuclear membrane. In this study, we also discovered that the PCYT1A gene expression level is significantly higher than the ERCC1 gene expression level in a certain population of lung cancer patient tissue samples. To develop the best monoclonal antibody for ERCC1 IHC analysis, 18 monoclonal antibodies were generated and 6 of them were screened against our protein microarray chip. Two clones showed high mono-specificity on the protein microarray chip test and both worked for the IHC application.
  
  
  CONCLUSIONS
  In summary, the 8F1 clone is not suitable for ERCC1 IHC assay due to its cross-reactivity with PCYT1A protein. Two newly generated monoclonal antibodies, 4F9 and 2E12, demonstrated ultra-specificity against ERCC1 protein and superior performance for IHC analyses.
authors.affiliation: OriGene Technologies Inc, 9620 Medical Center Drive, Rockville, MD 20850, USA. dma@origene.com
authors.forename: Donghui
authors.initials: D
authors.lastname: Ma
authors.forename: Dror
authors.initials: D
authors.lastname: Baruch
authors.forename: Youmin
authors.initials: Y
authors.lastname: Shu
authors.forename: Kehu
authors.initials: K
authors.lastname: Yuan
authors.forename: Zairen
authors.initials: Z
authors.lastname: Sun
authors.forename: Kaiyan
authors.initials: K
authors.lastname: Ma
authors.forename: Toan
authors.initials: T
authors.lastname: Hoang
authors.forename: Wei
authors.initials: W
authors.lastname: Fu
authors.forename: Li
authors.initials: L
authors.lastname: Min
authors.forename: Zhu-Sheng
authors.initials: ZS
authors.lastname: Lan
authors.forename: Fangxun
authors.initials: F
authors.lastname: Wang
authors.forename: Lori
authors.initials: L
authors.lastname: Mull
authors.forename: Wei-Wu
authors.initials: WW
authors.lastname: He
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology; D000918:Antibody Specificity; D014408:Biomarkers, Tumor / Q000276:immunology* / Q000378:metabolism; D002289:Carcinoma, Non-Small-Cell Lung / Q000737:chemistry / Q000378:metabolism; D019916:Choline-Phosphate Cytidylyltransferase / Q000276:immunology / Q000378:metabolism; D003429:Cross Reactions; D004268:DNA-Binding Proteins / Q000276:immunology* / Q000378:metabolism; D004720:Endonucleases / Q000276:immunology* / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D008175:Lung Neoplasms / Q000737:chemistry / Q000378:metabolism; D040081:Protein Array Analysis / Q000379:methods*
chemical_list: D000911:Antibodies, Monoclonal; D014408:Biomarkers, Tumor; D004268:DNA-Binding Proteins; D019916:Choline-Phosphate Cytidylyltransferase; C517213:PCYT1A protein, human; C071447:ERCC1 protein, human; D004720:Endonucleases