DESCRIPTION

PPIF, also known as Cyclophilin D (CypD), CYP3, Rotamase F, is a highly conserved peptidyl-prolyl cis-trans isomerase (PPIase) that plays an important role in mitochondrial biology. PPIF has been thought to regulate the opening of the permeability transition pore (PTP), a large conductance pore whose opening depolarizes the inner mitochondrial membrane (IMM) to decrease ATP and increase reactive oxygen species production. PPIF is a key mitochondrial target for Aβ-induced mitochondrial and synaptic dysfunction. Blockade of PPIF may be a potential therapeutic approach for halting Alzheimer's disease.

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	36.65	2.11	5.75
2	8	8.54	0.48	5.64
3	8	4.47	0.22	5.00

Inter-assay Precision

Sample	n	Mean (ng/mL)	SD	CV%
1	16	35.35	2.69	7.62
2	16	8.29	0.75	9.02
3	16	4.43	0.33	7.48

RECOVERY

The recovery of human/mouse PPIF spiked to three different levels throughout the range of the assay in lysate was evaluated.

Sample Type		Average% of Expected	Range (%)
Lysate	1:16	85	84-86

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. (The lysate was initially diluted 1:4.)

		Lysate
1:2	Average% of Expected	100
	Range (%)	-
1:4	Average% of Expected	90
	Range (%)	74-105
1:8	Average% of Expected	101
	Range (%)	87-114
1:16	Average% of Expected	111
	Range (%)	106-116

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human/mouse PPIF produced at Sunlong Medical™.