Development of an ELISA for measurement of BCAR1 protein in human breast cancer tissue.
BACKGROUND
High concentrations of breast cancer anti-estrogen resistance 1 (BCAR1) protein measured by Western blotting in primary breast tumor cytosols are associated with early disease progression and failure of tamoxifen therapy. The aim of the present study was to develop an ELISA to measure BCAR1 quantitatively in extracts of human breast cancer tissue.


METHODS
A recombinant fragment of BCAR1 (the human homolog of murine p130Cas) was produced in bacterial M15 cells, purified, and injected into chickens and rabbits. The generated antibodies were affinity-purified and used for the construction of an ELISA. After validation, the results obtained with the ELISA were compared with Western blot findings on primary breast tumors.


RESULTS
The detection limit the BCAR1 ELISA was 0.0031 microg/L, and the within-run imprecision (CV) was <20% at concentrations down to 0.004 microg/L. The within-run imprecision (CV) was 1.0-7.2%, and the between-run CV was 3.6-5.4%. There was no cross-reactivity with family member HEF1. The assay exhibited parallelism of results between serial dilutions and a mean recovery (range) of 96 (79-118)%.


CONCLUSIONS
The ELISA measures BCAR1 in human breast cancer cytosols with high sensitivity and specificity. The assay can be used to confirm and to quantitatively extend previous semiquantitative Western blot data on the prognostic and predictive value of BCAR1 in human breast cancer; it can also be applied for other diseases.
Department of Chemical Endocrinology, University Medical Center Nijmegen, Nijmegen, The Netherlands.
Nicolai
N
Grebenchtchikov
Arend
A
Brinkman
Simone P J
SP
van Broekhoven
Danielle
D
de Jong
Anneke
A
Geurts-Moespot
Paul N
PN
Span
Harry A
HA
Peters
Henk
H
Portengen
John A
JA
Foekens
C G J Fred
CG
Sweep
Lambert C J
LC
Dorssers
D000818:Animals; D000918:Antibody Specificity; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000737:chemistry* / Q000175:diagnosis; D045744:Cell Line, Tumor; D002645:Chickens; D050719:Crk-Associated Substrate Protein; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005260:Female; D006801:Humans; D011237:Predictive Value of Tests; D011379:Prognosis; D011506:Proteins / Q000032:analysis*; D011817:Rabbits; D050717:Retinoblastoma-Like Protein p130; D012680:Sensitivity and Specificity
C493841:BCAR1 protein, human; D050719:Crk-Associated Substrate Protein; D011506:Proteins; D050717:Retinoblastoma-Like Protein p130