Contamination detection assays can be used in a broad range of applications to measure contamination in a variety of sample content from fermentation supernatants and purification processes to biochemical preparations such as serum and other bodily fluids. They also measure the presence of host cell DNA as well as other DNA that might have been introduced during the biopharmaceutical manufacturing process.
Molecular Devices offers two contaminant detection assay kits under its Threshold brand: Threshold® Immunoligand Assay (ILA) and Threshold® Total DNA Assay
Threshold Immunoligand Assay measures a broad range of analytes such as proteins, peptides, sequence-specific DNA and microorganisms for biopharmaceutical development and production. Samples can range from fermentation supernatants, samples from a purification process, and other biochemical preparations to serum or other bodily fluids. Assays can be developed using a sandwich or competitive format depending on the size and characteristics of the analyte to be measured.
Threshold Total DNA Assay quantitatively measures picograms of single-stranded DNA. Unlike hybridization techniques which require DNA probes or primers with a specific nucleic acid sequence, the Threshold Total DNA Assay measures DNA with broad sequence specificity. This enables the Threshold System to measure host-cell DNA as well as other DNA that might have been introduced during the biopharmaceutical manufacturing process. In addition, since the assay does not require sequence-specific probes or primers, it can easily be applied to different samples purified from different host organisms.
Immunoligand Assay (ILA)
Total DNA Assay
In the reaction stage, the labeled antibodies, the sample containing the analyte, streptavidin and anti-fluorescein/urease conjugate provided in the ILA Detection Kit are incubated to form a reaction complex. Since this binding occurs in solution, all molecules are maintained in their native conformation to assure accurate measurement of the analyte and 100% activity of the antibodies. In similar fashion, a reaction complex is formed in the Total DNA Assay when biotinylated single-stranded binding protein and anti-ssDNA antibody, conjugated to urease, bind without sequence specificity to single-stranded DNA. All assay components are included in the complete Total DNA Assay Kit.
During the separation stage, the strong affinity of streptavidin for biotin is used to capture and concentrate the reaction complexes onto a biotinylated membrane by a filtration step. The Threshold workstation contains four 8-channel filtration units with the possibility of adding two auxiliary manifolds for a total simultaneous filtration of 96 samples.
In the detection stage, the captured reaction complex on the membrane is placed into the Threshold reader which contains the substrate urea and the light-addressable potentiometric sensor (LAPS). Inside the reader, urea is hydrolyzed by urease producing a pH change in a microvolume of less than 1 μl. Urease activity from eight different samples is simultaneously detected by the sensor during a 90 second kinetic measurement. The extreme sensitivity, precision and reproducibility of the Threshold® System is due to the combination of the small sample volume with the low-noise sensor.