An ELISA-based procedure for assaying proteins in digests of human leukocytes and cell lines, using specifically selected peptides and appropriate antibodies.
BACKGROUND
We describe the application of an ELISA-based assay (the Peptidomatrix) that can be used to simultaneously identify and quantitate a number of proteins in biological samples. The biological sample (blood component, biopsy, culture or other) is first lysed to release all the proteins, without any additional separation. The denatured proteins in the sample are then digested in bulk with the desired proteolytic enzyme(s). The peptides in the digest are then assayed by appropriate antibodies, using a competition ELISA protocol.


RESULTS
As an example of its use, the present paper applies the Peptidomatrix to the assay of four membrane proteins MDR1 (P-glycoprotein or ABCB1), MRP1 (ABCC1), BCRP/MXR (ABCG2) and the alpha subunit of the Na, K_ATPase (ATP1A1), present in a number of cell lines and in human lymphocytes. We show that we can detect and quantitate these proteins, using a series of peptide-antibody pairs, and that we can differentiate between cell lines or cell preparations that express the target proteins and those that do not.


CONCLUSIONS
We have devised a simple, ELISA-based proteomics assay that enables the quantitation of designated proteins in a cell or tissue sample, and that can be used in any laboratory, with minimal specialized equipment.
Braitbard
Ori
O
Biological Chemistry, Silberman Institute of Life Sciences, Hebrew University, Jerusalem, Israel.
Bishara-Shieban
Janette
J
MDR Tests Ltd, 28 Pierre Koenig St, Jerusalem, Israel.
Glickstein
Hava
H
Biological Chemistry, Silberman Institute of Life Sciences, Hebrew University, Jerusalem, Israel.
Kott-Gutkowski
Miriam
M
Biological Chemistry, Silberman Institute of Life Sciences, Hebrew University, Jerusalem, Israel.|MDR Tests Ltd, 28 Pierre Koenig St, Jerusalem, Israel.
Pace
Umberto
U
MDR Tests Ltd, 28 Pierre Koenig St, Jerusalem, Israel.
Rund
Deborah G
DG
Hematology Department of Hadassah University Hospital, Jerusalem, Israel.
Stein
Wilfred D
WD
Biological Chemistry, Silberman Institute of Life Sciences, Hebrew University, Jerusalem, Israel.|MDR Tests Ltd, 28 Pierre Koenig St, Jerusalem, Israel.