Human p53 ELISA Kit

Sample

Cell culture supernatant and serum

Species Reactivity

Human

Detection Method

sELISA

Intended Use

The p53 ELISA Kit is an enzyme-linked immunosorbent assay for the quantitative detection of human p53. For research use only. Not for diagnostic procedures.

Storage

Store kit reagents between 2°C and 8°C. Immediately after use remaining reagents should be returned to cold storage (2°C to 8°C). Expiry of the kit and reagents is stated on labels.
Expiry of the kit components can only be guaranteed if the components are stored properly, and if, in case of repeated use of one component, this reagent is not contaminated by the first handling.

Performance Characteristics

Sample Stability
a. Freeze-Thaw Stability
Aliquots of serum samples (spiked or unspiked) were stored at -20°C and thawed 5 times, and the human p53 levels determined. There was no significant loss of human p53 immunoreactivity detected by freezing and thawing up to 3 freeze-thaw cycles. A significant decrease of human p53 immunoreactivity was detected after 5 freeze-thaw cycles.
b. Storage Stability
Aliquots of serum samples (spiked or unspiked) were stored at –20°C, 2–8°C, room temperature, and at 37°C, and the human p53 level determined after 24 hours. There was no significant loss of human p53 immunoreactivity detected during storage under above conditions.

Precision

Intra-Assay
Reproducibility within the assay was evaluated in 3 independent experiments. Each assay was carried out with 6 replicates of 7 serum samples containing different concentrations of human p53. 2 standard curves were run on each plate. Data below show the mean human p53 concentration and the coefficient of variation for each sample (see Table). The calculated overall intra-assay coefficient of variation was 5.5 %.
Table: The mean human p53 concentration and the coefficient of variation for each sample

Inter-Assay
Assay to assay reproducibility within one laboratory was evaluated in 3 independent experiments. Each assay was carried out with 6 replicates of 7 serum samples containing different concentrations of human p53. 2 standard curves were run on each plate. Data below show the mean human p53 concentration and the coefficient of variation calculated on 18 determinations of each sample (see Table). The calculated overall inter-assay coefficient of variation was 8.9 %.
Table: The mean human p53 concentration and the coefficient of variation of each sample

Sensitivity

The limit of detection of human p53 defined as the analyte concentration resulting in an absorbance significantly higher than that of the dilution medium (mean plus 2 standard deviations) was determined to be 0.33 U/mL (mean of 6 independent assays).

General Description

The p53 protein is highly conserved the evolution and expressed in most normal tissues. Wild-type p53 has been shown to be a sequence- specific transcription factor, directly interacting with various cellular and viral proteins.
p53 is considered a stress response gene, the p53 protein acts to induce cell cycle arrest or apoptosis in response to DNA damage, thereby maintaining genetic stability in the organism. These functions are executed by a complex and incompletely understood series of steps known as the p53 pathway.
p53 is a tumor suppressor gene which induces apoptosis. An inverse relationship in some neoplasms has been shown between p53 and Bcl-2, a proto-oncogene inhibiting apoptosis.
The Bcl-2 homologue BAX gene has also been characterized to be regulated by p53; BAX acts to accelerate the rate at which apoptosis proceeds.
p53 was further shown to mediated apoptosis through cell surface trafficking of APO-1/Fas.
p53 as a transcription factor induces the expression of p21WAF1/CIP1/Sdi1 leading to G1 arrest of the cell. p53 is known to be regulated by phosphorylation by a number of specific protein kinases. Activation by autoproteolysis has been shown.
In addition to proliferation control, a role for p53 is cell senescence has been described.
More than 500 mutations in the p53 gene have been described. These mutations were found in various types of malignancies, hematologic as well as solid tumors. However, all the mutants are not necessarily equivalent in terms of biological activity.

Standard Curve

A representative standard curve is shown in Figure. Note: Do not use this standard curve to derive test results. Each laboratory must prepare a standard curve for each group of microwell strips assayed.

Typical data using the p53 ELISA
Measuring wavelength: 450 nm
Reference wavelength: 620 nm

The OD values of the standard curve may vary according to the conditions of assay performance (e.g., operator, pipetting technique, washing technique, or temperature effects). Furthermore, shelf life of the kit may affect enzymatic activity and thus color intensity. Values measured are still valid.