The Threshold System
Frequently Asked Questions
What does the Threshold® System do?
The Threshold system is an analytical system that was designed specifically to measure contaminants in
biopharmaceuticals, including total DNA and proteins, such as host cell proteins, bovine contaminants (BSA, IgG,
transferrin and insulin), and Proteins A and G. Recently new applications have been developed for measuring
sequence-specific DNA and plasmid copy number.
What are the advantages of the Threshold System?
The key advantages are:
- A wide range of analytes can be measured using this one complete system. These include peptides to large proteins, non sequence-specific total DNA, and sequence-specific DNA.
- The initial binding or hybridization of the analyte occurs in liquid phase, which maintains the native conformation of the proteins and allows faster binding/hybridization kinetics.
- A silicon sensor is used for detection. The design of this sensor allows excellent sensitivity with high precision and reproducibility.
- Faster assays are possible because shorter incubations are required, and detection is completed in minutes.
How does the Threshold System work?
The sample containing the analyte of interest is incubated with the appropriate binding proteins or oligonucleotide
probes. The analyte will be bound by the binding proteins or hybridized to the probes to form a complex. The sample
mixture is then filtered through a membrane where the analyte complex is captured and separated from the sample. The
captured analyte complex will contain enzyme proportional to the amount of complexed analyte. The membrane is inserted
into the Threshold reader which contains the silicon sensor. A kinetic measurement of the enzyme activity is completed
in 90 seconds. This data is then processed by the Threshold software and quantitation is provided. Please see The
Immuno-Ligand and Total DNA Assay Principles pages for greater detail.
What is the sensitivity of Threshold System assays?
The sensitivity depends on the analyte being measured. For total DNA, using the Total DNA Assay Kit, the limit of
detection is 2 picograms. When measuring contaminant proteins using the Immuno-Ligand Assay Kit, the limit of
detection will be dependent upon the antibodies used. Molecular Devices has prepared Applications Notes for many
of the common contaminants. As examples, the limit of detection for BSA is 125 pg/mL, for bovine IgG it is 50 pg/mL,
and for Protein A it is 20 pg/mL (in the presence of MuIgG1, the limit of detection is 25 ppb). Using the
Immuno-Ligand Assay Kit and a model system developed for measuring the E. coli plasmid pGEM3, the limit of detection
is 400 attomoles (which is equivalent to 750 pg or 235 x 106 molecules). For each specific analyte, the limit of
detection is dependent upon the binding proteins, or DNA probes, and the assay configuration selected. However, the
Threshold system will allow the maximum sensitivity with the available reagents, without the use of radioactivity.
How is high sensitivity achieved without using radioactivity?
After the analyte is either bound by the binding proteins or hybridized to the DNA probes, the sample is filtered
through a membrane where the formed complex is actively captured and concentrated. This membrane is then transferred
to the detector where the enzyme activity of a high turnover enzyme is measured in less than 1 µl volume per
sample.
What is provided in the reagent kits?
The Total DNA Assay Kit includes all of the reagents required to test a sample. This includes a calf thymus DNA
"High Calibrator" for preparing the standard curve, a DNA free "Zero Calibrator" that can be used for diluting the
sample and preparing the standards, a "Positive Control" for monitoring the accuracy of the standard curve preparation,
the "Labeling Reagent" that will bind to the sample DNA, and the membrane sticks for capturing the bound DNA. A
substrate concentrate is also included for detection, as well as the appropriate buffer and wash solutions.
The Immuno-Ligand Assay Kit includes all of the reagents required to perform a Threshold system assay, except the
binding proteins (such as antibodies) or DNA probes specific for the analyte of interest. The kit reagents include
"Capture Reagent," "Enzyme Reagent," membrane sticks, substrate concentrate, and appropriate buffers and washes. A
"Universal Standard" is also included, which can be used for training and trouble-shooting purposes without using
the specific binding proteins and analyte.
How long does it take to complete an assay?
The Total DNA Assay, excluding any required sample pretreatment, is completed in less than two hours. For an
Immuno-Ligand Assay, the incubation time will be dependent upon the binding proteins or probes selected and the
assay configuration. For example, the BSA assay is completed in 3 1/2 hours, and the bovine IgG and Protein A assays
are completed in 3 hours. If maximum sensitivity is not required, the incubation can be reduced to less than 15
minutes, which allows a total assay time of less than 1 hour. The specific-sequence DNA assay, after restriction
digestion of the sample DNA, requires as little as 60 minutes for hybridization and quantitation.
Is the Threshold System approved by the FDA?
The FDA does not approve or disapprove of commercial assay systems. However, there have been many IND's filed with
the FDA that have used the Threshold Total DNA Assay for measuring contaminant DNA, or the Immuno-Ligand Assay for
measuring Host Cell Proteins or other protein contaminants, and all have proceeded to clinical trials with no
questions that were transmitted to Molecular Devices. There have also been PLAs submitted and approved that have
included Threshold Total DNA Assay data and/or Immuno-Ligand Assay data for process validation and/or final QC
release. Biopharmaceutical products have been approved in Europe that were validated and/or released with Total
DNA and/or Immuno-Ligand Assay data.
How Long has this system been in use?
The first Threshold was shipped in January of 1989.
Who are the primary customers for the Threshold System?
Over 300 Threshold systems have been sold and are currently in use at most of the leading-edge biopharmaceutical
and pharmaceutical companies worldwide.
Is the Threshold System sold and supported worldwide?
Molecular Devices currently has technical support centers in Sunnyvale, California, and Munich, Germany. There are
also field service offices located in Sunnyvale and Boston, Massachusetts; London, England; and Japan. Direct sales
offices are located throughout the US, in Germany and the United Kingdom and Japan. The remaining world areas are
handled by Molecular Devices distributors.
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