Limits...
Using protein microarray technology to screen anti-ERCC1 monoclonal antibodies for specificity and applications in pathology.

Ma D, Baruch D, Shu Y, Yuan K, Sun Z, Ma K, Hoang T, Fu W, Min L, Lan ZS, Wang F, Mull L, He WW - BMC Biotechnol. (2012)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: OriGene Technologies Inc, 9620 Medical Center Drive, Rockville, MD 20850, USA. dma@origene.com

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.

Conclusion: 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.

Show MeSH

Related in: MedlinePlus

qPCR analysis of mRNA expression profile for ERCC1 and PCYT1A genes on 24 lung cancer patient tissues. A. qPCR standard. The experimental data using OriGene’s ERCC1 and PCYT1A qPCR standards. A formula for copy number calculation is shown on the chart. B. qPCR lung cancer tissue expression analysis. qPCRdata on OriGene’s TissueScan lung cancer cDNA array panel (HLRT104). The data were grouped based on cancer patient stages.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3526464&req=5

Figure 5: qPCR analysis of mRNA expression profile for ERCC1 and PCYT1A genes on 24 lung cancer patient tissues. A. qPCR standard. The experimental data using OriGene’s ERCC1 and PCYT1A qPCR standards. A formula for copy number calculation is shown on the chart. B. qPCR lung cancer tissue expression analysis. qPCRdata on OriGene’s TissueScan lung cancer cDNA array panel (HLRT104). The data were grouped based on cancer patient stages.

Mentions: Quantitative real-time PCR can provide direct measurements of mRNAs level for different genes in the same tumor tissue sample. To ensure PCR amplification occurs at the same efficiency and to obtain an absolute mRNA copy number in the tissue mRNA samples, OriGene’s gene-specific qPCR copy number standards were serial-diluted and used for CT value measurement. The final data are summarized and plotted in Figure5A. Our data show that the gene-specific probes chosen for ERCC1 and PCYT1A exhibit similar amplification efficiencies with their target genes. A copy number calculation formula was also created based on the standard curves.


Using protein microarray technology to screen anti-ERCC1 monoclonal antibodies for specificity and applications in pathology.

Ma D, Baruch D, Shu Y, Yuan K, Sun Z, Ma K, Hoang T, Fu W, Min L, Lan ZS, Wang F, Mull L, He WW - BMC Biotechnol. (2012)

qPCR analysis of mRNA expression profile for ERCC1 and PCYT1A genes on 24 lung cancer patient tissues. A. qPCR standard. The experimental data using OriGene’s ERCC1 and PCYT1A qPCR standards. A formula for copy number calculation is shown on the chart. B. qPCR lung cancer tissue expression analysis. qPCRdata on OriGene’s TissueScan lung cancer cDNA array panel (HLRT104). The data were grouped based on cancer patient stages.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3526464&req=5

Figure 5: qPCR analysis of mRNA expression profile for ERCC1 and PCYT1A genes on 24 lung cancer patient tissues. A. qPCR standard. The experimental data using OriGene’s ERCC1 and PCYT1A qPCR standards. A formula for copy number calculation is shown on the chart. B. qPCR lung cancer tissue expression analysis. qPCRdata on OriGene’s TissueScan lung cancer cDNA array panel (HLRT104). The data were grouped based on cancer patient stages.
Mentions: Quantitative real-time PCR can provide direct measurements of mRNAs level for different genes in the same tumor tissue sample. To ensure PCR amplification occurs at the same efficiency and to obtain an absolute mRNA copy number in the tissue mRNA samples, OriGene’s gene-specific qPCR copy number standards were serial-diluted and used for CT value measurement. The final data are summarized and plotted in Figure5A. Our data show that the gene-specific probes chosen for ERCC1 and PCYT1A exhibit similar amplification efficiencies with their target genes. A copy number calculation formula was also created based on the standard curves.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: OriGene Technologies Inc, 9620 Medical Center Drive, Rockville, MD 20850, USA. dma@origene.com

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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus