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Technology Overview

 

Z–TECT™” for “Zeptomole–deTECTion” with “zepto” being the prefix (abbreviated “z”) in the International System of Units denoting a factor of 10E–21 (following “atto (a)” for 10E–18, “femto (f)” for 10E–15, “pico (p)” for 10E–12 and “nano (n)” for 10E–9).

 

In contrast with the current sensitivity–limited immunoassays, Akrivis’ Z–TECT™ is a unique technology platform which provides accurate and reproducible detection and quantification of analytes from various sample types down to the zeptomole range (10E–21 moles), allowing the detection of less than a thousand individual molecules. This achievement represents a 10–100 million–fold improvement in sensitivity over the best ELISA (Enzyme–Linked Immuno Sorbent Assay) immunoassays which are currently limited to 10E–13 to 10E–14 moles.

 

In essence, Z–TECT™ is a signal amplification system composed of a Targeting Unit and a Signaling Unit. The Z–TECT™ Signaling Unit provides many–fold enhancement of the detection signal for each binding event between the Targeting Unit and the targeted antigen/biomolecule. In addition, since the system is specially designed to maintain a low background level, the resulting signal–to–noise ratio is increased and better quantitative accuracy can be achieved.

 

Results described below demonstrate the ultrasensitive detection of trace amounts of analytes present in biological samples from human sera obtained with Z–TECT™:

  • Targeted antigens aer soluble cardiac Myosin Heavy Chain fragments (cMHCf) in sera of patients obtained at the time of emergency room admission for acute myocardial infarction (AMI).
  • Detection antibody is anti–myosin;
  • Signaling molecule is Horse Radish Peroxidase (HRP) and chromogen can be orthophenyldiamine or K–Blue;
  • Signal output is Optical Density (OD, 490nm), read with a standard ELISA reader.

 

Sensitivity was assessed in vitro with a controlled experiment in which the assay was performed with increasingly sensitive Z–TECT™ Signaling Units and compared to a conventional competitive inhibition ELISA standard curve. Increasing sensitivity of the standard curves was obtained with increasing HRP–loading of the Z–TECT™ Signaling Units (Figure 1).


In Figure 1, the X axis represents the original antigen concentration in the solution from which 50 μl aliquots were taken for the assays. 50 μl aliquots of 100 μg/ml canine cardiac myosin contained 1x10E–11 moles and 50 μl aliquots of 1x10E–8 μg/ml solution contained 1x10E–21 moles. Sensitivity increased progressively from 1x10E–13 moles of antigen for the standardELISA (green circles) to 1x10E-21 moles for Z–TECT™ with the most sensitive Signaling Unit #5 (purple circles).


 

Table 1 below compares the calculated sensitivities and number of individual molecules detected between a conventional ELISA and Z–TECT™ with increasingly sensitive Signaling Units.

ZEPTACSYS™ Signaling Units

ELISA

#1

#2

#3

#4

#5

Ag concentration at -2SD of max.
AB binding (ug/ml)
  1.2x10-2 1.2x10-5 1.1x10-6 1.2x10-7 1.5x10-8
Sensitivity (Moles) 1.1x10-13 1.2x10-15 1.2x10-18 1.1x10-19 1.2x10-20 1.5x10-21
Number of Molecules 6.62x1010 7.23x108 7.23x105 6.62x104 7.23x103 903

At its maximum sensitivity of 1.5x10E–21 moles, Z–TECT™ detected 903 individual cMHCf molecules compared to the 66.2 billion molecules required for detection by conventional ELISA. This indicates a 75 million–fold increase in sensitivity from ELISA to Z–TECT™.

 

Additional Features

In addition to Z–TECT™’s ultrasensitivity, or when such ultrasensitivity is not required, the following features and design variations can provide further advantages:

  • Substitution of HRP with other signaling molecules such as rhodamine, green fluorescent protein (GFP), Fluorescein, quantum dots for multiplexing, any fluorophore or any bioluminescence generating system;
  • All Z–TECT™ benefits can apply to other immunoassays such as Western Blot, flow cytometry, immunohistochemistry, immunocytochemistry and radioimmunoassays;
  • Substitution of HRP with radiolabels such as Technetium–99m or Indium–111 resulting in high contrast in vivo imaging applications which benefit from combining enhanced signal with low background level;
  • Substitution of HRP with drug molecules such as doxorubicin,1 potentially resulting in safer and more efficacious targeted drug delivery systems.