Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® EGF Receptor (L858R Mutant Specific) Sandwich ELISA Kit #8951

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Kit Includes Volume Solution Color
EGF Receptor (L858R Mutantl Specific) Rabbit Antibody coated microwells 96 tests
EGF Receptor Detection Ab 11 ml Green
Anti-mouse IgG, HRP-linked Antibody 11 ml Red
TMB Substrate #7004 11 ml Colorless
STOP Solution #7002 11 ml Colorless
Sealing Tape 2 sheets
ELISA Wash Buffer (20X) 25 ml Colorless
ELISA Sample Diluent 25 ml Blue
Cell Lysis Buffer (10X) #9803 15 ml Yellowish

Note: 12 8-well modules –Each module is designed to break apart for 8 tests.
Note: Kit should be stored at 4°C with the exception of Cell Lysis Buffer, which is stored at –20°C (packaged separately).

Species Cross-Reactivity

H

Reactivity Key:  H=Human
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Description

The PathScan® EGF Receptor (L858R Mutant Specific) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of EGF receptor L858R mutant protein. An EGF receptor (L858R Mutant Specific) rabbit mAb has been coated on the microwells. After incubation with cell lysates, EGF receptor L858R mutant protein is captured by the coated antibody. Following extensive washing, an EGF receptor mouse monoclonal detection antibody is added to detect captured EGF receptor mutant protein. Anti-mouse IgG, HRP-linked antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of the absorbance for this developed color is proportional to the quantity of EGF receptor L858R mutant protein.Antibodies in kit are custom formulations specific to kit.

Specificity / Sensitivity

PathScan® EGF Receptor (L858R Mutant Specific) Sandwich ELISA Kit #8951 detects endogenous levels of EGF receptor L858R mutant protein in human cells, as shown in Figure 1. The kit sensitivity is shown in Figure 2. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.

ELISA - Western correlation

ELISA - Western correlation

Figure 1. EGFR mutant protein from H3255 cells can be detected by PathScan® EGF Receptor (L858R Mutant Specific) Sandwich ELISA Kit #8951. However, this kit cannot detect wild type EGFR protein from A-431 cells or EGFR E746-A750del protein from HCC827 cells. EGFR (either wild type or mutant) from these three cell lines can be detected by PathScan® Total EGF Receptor Sandwich ELISA Kit #7250, but no detection is found in MCF7 cells, which lack expression of EGFR. The absorbance readings at 450 nm are shown in the top figure, while the corresponding western blots using EGF Receptor (L858R Mutant Specific) (43B2) Rabbit mAb #3197, EGF Receptor (E746-A750del Specific) (6B6) XP® Rabbit mAb #2085, or EGF Receptor (C74B9) Rabbit mAb #2646 are shown in the bottom figure.

Sensitivity

Sensitivity

Figure 2. The relationship between protein concentration of lysates from A-431, H3255, or HCC827 cells and the absorbance at 450 nm is shown. A-431, H3255, or HCC827 cells (85% confluence) were harvested and then lysed.

Background

The epidermal growth factor (EGF) receptor is a 170 kDa transmembrane tyrosine kinase that belongs to the HER/ErbB protein family. Research studies have shown that somatic mutations in the tyrosine kinase domain of EGF receptor (EGFR) are present in a subset of lung adenocarinomas that respond to EGFR inhibitors, such as gefinitib and erlotinib (1-3). Two types of mutations account for approximately 90% of mutated cases: a specific point mutation, L858R, that occurs in exon 21 and short in-frame deletions in exon 19 (4,5). The most frequent exon 19 deletion is E746-A750, accounting for 90% of lesions at this site, although some rare variants occur.

  1. Lynch, T.J. et al. (2004) N Engl J Med 350, 2129-39.
  2. Pao, W. et al. (2004) Proc Natl Acad Sci USA 101, 13306-11.
  3. Haber, D.A. et al. (2005) Cold Spring Harb Symp Quant Biol 70, 419-26.
  4. Kosaka, T. et al. (2004) Cancer Res 64, 8919-23.
  5. Riely, G.J. et al. (2006) Clin Cancer Res 12, 7232-41.

Application References

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For Research Use Only. Not For Use In Diagnostic Procedures.

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