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.
Two contaminant detection assay kits are available under the 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.
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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.
Please click here to enlarge image.
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.
Application Note
The FDA recommends that all quality control tests used for biopharmaceutical product release follow current Good Manufacturing Practices (cGMP's) outlined in the Code of Federal…
Application Note
A DNA extraction method was evaluated using a commercially available extraction kit for pretreatment of biopharmaceutical samples to be tested in the Threshold® Total DNA Assay. This…
Application Note
Measurement of specific DNA sequences can be achieved using the Threshold® System. Biotinylated and fluoresceinated oligonucleotide probes specific to the target sequence to be…
Application Note
Protein G, purified from the cell wall of Group G Streptococci, is a 30,000-35,000 Dalton protein which binds to the Fc region of antibodies1 . This characteristic is utilized for the…
Application Note
Protein A, purified from the cell wall of S. aureus, is a 42,000 Dalton protein. It has four binding sites for the Fc region of antibodies of which only two sites may be occupied…
Application Note
The Threshold® Immuno-Ligand Assay (ILA) adapts reagents used in ELISA, RIA or radioreceptor assays for the Threshold System by covalently attaching biotin or fluorescein labels to the…
Application Note
Human transferrin is an 80,000 Dalton glycoprotein found in human serum that facilitates transport of iron between cells. The iron-poor form, apo-transferrin, combines with the Fe2+…
Application Note
Polypeptides other than the biological product of interest that are produced by the host cell during cell culture or fermentation (“host cell-derived protein” or “HCP”) can cause an…
Application Note
Bovine serum albumin (BSA), a 63,000 Dalton protein, is a major constituent of bovine serum. Fetal calf serum containing BSA can be used in mammalian cell culture media for the…
Application Note
Bovine transferrin, a 76,000 Dalton glycoprotein, is one of the constituents of bovine serum. Transferrin found in serum can be associated with iron ions. The iron-saturated form is…
Application Note
Bovine IgG, a 160,000 Dalton protein, is one of the constituents of bovine serum. Fetal calf serum containing bovine IgG is commonly used in mammalian cell culture media for the…
Application Note
Accurate measurement of the concentration of a biological product is important in bioreactor/fermenter monitoring, downstream processing and final product quality control. Currently,…
Application Note
The Threshold Total DNA Assay quantitates contaminant DNA in biopharmaceutical drugs. DNase I can be used in conjunction with the Total DNA Assay for two purposes: To validate that the…
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Our highly-qualified teams are on the frontlines with our customers, conducting remote or on-site product demonstrations, webinars, and more to help you solve your tough research challenges. How can we help you today?
I’d like to…