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Immunoassays | Prototype

Erenna® tAKT1 Immunoassay Prototype Assay (Cat# 03‐0036‐AA)

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Introduction

The Erenna® tAKT1 Immunoassay Evaluation Reagent Kit uses a quantitative fluorescent sandwich immunoassay technique to measure tAKT1 in human cell lysate samples. A capture antibody specific for tAKT1 has been pre-coated onto paramagnetic microparticles (MP). The user pipettes MP, standards, and samples into uncoated microplate wells. During incubation, the tAKT1 present in the sample binds to the capture antibody on the coated MP. Unbound molecules are washed away during the subsequent buffer exchange and wash steps. Fluor-labeled detection antibody is added to each well and incubated. This detection antibody recognizes and binds to tAKT1 that has been captured onto the MP. During the following wash step the MPs are transferred to a clean plate. Elution buffer is then added and incubated. The elution buffer dissociates the bound protein sandwich from the MP surface, releasing the labeled antibodies. These antibodies are separated during transfer to a final microplate. The plate is loaded into the Erenna System where the labeled molecules are detected and counted. The number of fluor-labeled detection antibodies counted is directly proportional to the amount of tAKT1 present in the sample when captured. The amount of tAKT1 in unknown samples is interpolated from a standard curve.

Assay Performance


TABLE 1. Analytical sensitivity of the Erenna® tAKT1 Immunoassay Prototype Assay1
Lower Limit of Detection 1.4 pg/mL
Lower Limit of Quantification2 12.3 pg/mL
Upper Limit of Quantification 1000 pg/mL
Low-end CV% Range 3 - 14%
Low-end CV% Average 9%
Recommended Sample Volume 100 μL

1 See product insert for updated values
2 LLoQ = 20% CV and ± 20% recovery


FIGURE 1. Mean endogenous [tAKT1] measured in matched normal, metastatic and primary tumor tissue samples from four individual patients. The Erenna® tAKT1 Immunoassay Evaluation Reagent Kit is able to quantify [tAKT1] elevations in metastatic and tumor tissues compared to adjacent normal tissue, demonstrating the sensitivity needed to accurately quantify endogenous tAKT1 in clinically relevant tissue types.

FIGURE 2. Erenna® tAKT1 Immunoassay Evaluation Reagent Kit assay low-end standard curve signal (left) and curve fit (right).

Representative data shown for demonstration purposes only. Individual results may vary depending upon samples tested and protocol used.

Biology and Disease

The serine-threonine protein kinase AKT family is the primary downstream mediator of the phosphatidylinositol-3-kinase (PI3K) signaling pathway. Members of the family (AKT1, AKT2, and AKT3) require two separate kinases for complete activation. AKT1 phosphorylates mTOR, whose downstream targets include other kinases, cell cycle regulators, and transcription factors that are essential for many cell proliferation and survival processes. AKT1 also up-regulates matrix metalloproteinases and is thus implicated in tumor progression. AKT1 and other members of the AKT family have been an important focus in therapeutic development with their broad eff ects on the PI3K pathway.


TABLE 2. Additional UniProtKB/Swiss-Prot Information*
Protein Name: RAC-alpha serine/threonine-protein kinase
UniProtKB/Swiss-Prot ID: P31749
Protein-protein interaction database: P31749
Alternative Names: Protein kinase B, Proto-oncogene c-Akt, RAC-PK-alpha
Gene Names: AKT1
Synonym Gene Names: PKB, RAC
Function: Plays a role as a key modulator of the AKT-mTOR signaling pathway controlling the tempo of the process of newborn neurons integration during adult neurogenesis, including correct neuron positioning, dendritic development and synapse formation.
Biological Processes: activation of pro-apoptotic gene products, negative regulation of protein kinase activity, regulation of translation
Molecular Function: protein serine/threonine kinase activity

* Source Info from www.uniprot.org.


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