Immunoassays | Prototype

Erenna® A?-40 Immunoassay Prototype Assay (Cat# 03-0020-AA)

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Introduction

The Erenna® A?-40 Immunoassay Evaluation Reagent Kit uses a quantitative fluorescent sandwich immunoassay technique to measure A?-40 in plasma and serum samples. A capture antibody specific for A?-40 has been pre-coated onto paramagnetic microparticles (MP). The user pipettes MP, standards, and samples into uncoated microplate wells. During incubation, the A?-40 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 A?-40 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 A?-40 present in the sample when captured. The amount of A?-40 in unknown samples is interpolated from a standard curve.

Assay Performance


TABLE 1. Analytical sensitivity of the Erenna® A?-40 Immunoassay Prototype Assay1
Lower Limit of Detection 5.9 pg/mL
Lower Limit of Quantification2 5.9 pg/mL
Upper Limit of Quantification 750 pg/mL
Low-end CV% Range 2 – 10%
Low-end CV% Average 6%
Recommended Sample Volume 100 ?L
Minimum Sample Volume Required3 10 ?L

1 See product insert for updated values
2 LLoQ = 20% CV and ± 20% recovery
3 based upon median [A?-40] in a healthy reference


FIGURE 1. Normal [A?-40] in 10 EDTA plasma donors, with median and interquartile range. The Erenna® A?-40 Evaluation Reagent Kit reliably quantifies A?-40 in healthy subjects, who have a median [A?-40] of 69.6 pg/mL that is well above the detection limit of 5.9 pg/mL.

FIGURE 2. Erenna® A?-40 Evaluation Reagent Kit 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

Amyloid beta proteins (40 and 42 amino acids) are the main constituent of amyloid plaques in the brains of Alzheimer?s disease (AD) patients. In healthy and disease states A?-40 is the more common form of the two (10?20X higher than A?-42) in both cerebrospinal fl uid (CSF) and plasma. In patients with AD, A?-42 primarily aggregates and deposits in the brain forming plaques. Thus the eff ective concentration of A?-42 monomer is decreased in the CSF of many AD patients. Recent studies suggest that a decrease in A?-42 concentrations (with a paralleled change in the ratio of A?-40/A?-42) can be monitored and may indicate AD progression.


TABLE 2. Additional UniProtKB/Swiss-Prot Information*
Protein Name: Amyloid beta A4 protein
UniProtKB/Swiss-Prot ID: P05067
Protein-protein interaction database: P05067
Alternative Names: ABPP, APPI
Gene Names: APP
Synonym Gene Names: A4, AD1
Function: Functions as a cell surface receptor and performs physiological functions on the surface of neurons relevant to neurite growth, neuronal adhesion and axonogenesis. Involved in cell mobility and transcription regulation through protein-protein interactions.
Biological Processes: neuron apoptosis, axon midline choice point recognition, Notch signaling pathway
Molecular Function: acetylcholine receptor binding, heparin binding

* Source Info from www.uniprot.org.


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