DESCRIPTION

LIM kinase-2 belong to a small subfamily with a unique combination of 2 N-terminal LIM motifs and a C-terminal protein kinase domain. LIMK2 is an important regulator of growth and invasion of several cancers, including pancreatic cancer, bladder cancer, osteosarcoma, and glioblastoma. LIMK2 regulates actin cytoskeletal reorganization through phosphorylating and inactivating cofilin, an actin-depolymerizing factor of actin filaments. It also may associate with gamma-tubulin and play a role in mitotic spindle assembly. Some studies have shown that LIMK2 expression and activity increases after treatment with anticancer drugs, and these studies have implicated LIMK2 in drug resistance.

PRINCIPLE OF THE ASSAY

Sandwich ELISA structure (Detection antibody labeled with HRP) A capture antibody is pre-coated onto the bottom of wells which binds to analyte of interest. A detection antibody labeled with HRP also binds to the analyte. TMB acts as the HRP substrate and the solution color will change from colorless to blue. A stop solution containing sulfuric acid turns solution yellow. The color intensity is proportional to the quantity of bound protein which is measurable at 450 nm with the correction wavelength set at 630 nm.

PRECISION

Intra-assay Precision (Precision within an assay) Three samples of known concentration were tested 8 times on one plate to assess intra-assay precision. Inter-assay Precision (Precision between assays) Three samples of known concentration were tested in 16 separate assays to assess inter-assay precision.

Intra-assay Precision

Sample	n	Mean (ng/mL)	SD	CV%
1	8	47.23	1.48	3.14
2	8	12.98	0.35	2.70
3	8	7.48	0.50	6.63

Inter-assay Precision

Sample	n	Mean (ng/mL)	SD	CV%
1	16	47.17	2.36	5.01
2	16	13.19	0.61	4.60
3	16	7.58	0.41	5.46

RECOVERY

The recovery of human LIMK2 spiked to three different levels throughout the range of the assay in cell lysate was evaluated.

Sample Type		Average% of Expected	Range (%)
Cell lysate	1:16	78	72-85

LINEARITY

To assess the linearity of the assay, samples were diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay.

		Cell lysate
1:2	Average% of Expected	100
	Range (%)	-
1:4	Average% of Expected	82
	Range (%)	72-90
1:8	Average% of Expected	90
	Range (%)	73-101
1:16	Average% of Expected	107
	Range (%)	91-126

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human Human LIMK2 Sandwich ELISA Kit produced at Sunlong Medical™.