DESCRIPTION

Transcription factor EB(TFEB) is one of the transcription factors that contain a basic HLH-ZIP domain(for DNA binding ) and HLH and ZIP domains( for loigomerization). TFEB specifically recognizes and binds to E-box sequences, which required dimerization with itself or another MiT/TFE family member. It plays a role in T-cell-dependent antibody responses to activated CD4+ T-cells and thymus-dependent humoral immunity once associated with TFE3. It also activates lysosomal gene by binding the CLEAR-box sequence in the regulatory region. It involves in the signal transduction processes required for normal vascularization of the placenta. The calculated molecular weight of TFEB is 53 kDa, but modified TFEB is about 65-70 kDa. TFEB mainly present in the cytoplasm. Under aberrant lysosomal storage conditions, it translocates from the cytoplasm to the nucleus. Fisetin increases the levels of TFEB localized in nuclei.

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	14.89	0.60	4.04
2	8	3.77	0.16	4.22
3	8	1.04	0.05	4.66

Inter-assay Precision

Sample	n	Mean (ng/mL)	SD	CV%
1	16	14.91	0.61	4.12
2	16	3.84	0.15	3.97
3	16	1.05	0.04	4.02

RECOVERY

The recovery of human TFEB 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	74	74-75
	1:32	88	82-100

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 cell lysate was initially diluted 1:2.)

		Cell lysate
1:2	Average% of Expected	100
	Range (%)	-
1:4	Average% of Expected	104
	Range (%)	103-104
1:8	Average% of Expected	103
	Range (%)	102-104
1:16	Average% of Expected	101
	Range (%)	99-104

CALIBRATION

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