Frequently Asked Questions
1. Can the kit be used to measure levels of Primate IFNβ
Yes. Human and primate IFN are very closely related and will activate our reporter gene.
2. What type of human cell is used in the kit?
The cell line used as the "reporter cell" has been designated "PIL5" and is derived from the U937 cell line which can be obtained from public sources as ATCC # CRL-1593.2. These cells have been characterized as human pro-monocytic with the constitutive expression of major histocompatibility complex (MHC) Class II antigens on the cell surface; in particular, the human lymphocyte antigen designated HLA-DR.
3. What is the reporter gene?
The reporter gene is firefly luciferase which is under the genetic control mechanism of the Interferon Sensitive Response Element (ISRE) from Interferon Sensitive Gene 15 (ISG15). These genetic components have been inserted "upstream" of a minimal SV40 promoter.
4. What type of luminometer is needed?
Any good instrument that has the capability to read glow luminescence in a 96-well plate format is suitable. The luciferase substrate system is not designed for use with the automated reagent injectors integrated into some luminometers.
5. What are the storage requirements?
At minus 80 degrees C
6. How does this test compare to ELISA?
The most important difference is that iLite measures bioactivity and not simply protein mass. An ELISA test can measure all IFN protein, including inactive molecules and some degradation products. iLite only measures IFN molecules that interact with the cell receptor and induce biological activity. The iLite test is also easier to use and less influenced by non-specific proteins in human serum.
7. How does this test compare to traditional BioAssay?
We have performed a Performance Evaluation Study to demonstrate that the iLite test has an excellent correlation in the determination of International Units using the conventional bioassay (i.e., viral interference using MDBK cells and VSV).
The iLite test is more sensitive (i.e., can measure less than 1 (one) IU in a ml of sample), requires much less time and demonstrates good intra- and inter-lot variation in repeatability and reproducibility. The iLite test is also easier to use.
8. How does a gene reporter assay work?
The interaction of the IFN in the sample with the IFN receptor on the "reporter cell" initiates a cascade of biosynthetic reactions (e.g., phosphorylation of signal proteins) that results in the induction of some 130 IFN sensitive genes. One of the IFN sensitive genes has been replaced with the gene for the enzyme "firefly luciferase".
Hence, in the presence of IFN in the sample, the luciferase enzyme is produced. The amount of luciferase enzyme produced over time is directly proportional to the amount of IFN in the sample. After the required incubation, the cells are broken open, substrates for the enzyme are added and the luminescence is determined.
9. How long does it take to complete a test?
After the kit has been removed from the freezer and thawed, the results can be available in 4 to 18 hours depending on the objectives of the user. If the expected level of IFN in the sample is greater than 5 IU/ml, a 6-10 hour incubation should be sufficient.
10. How can I get results quickly?
Basically, by shortening the incubation time. At high levels of IFN in the sample, the incubation time can be reduced proportionately. Studies have indicated that incubations times as short as 4 hours can result in reliable results with relatively high concentrations of IFN ( i.e., greater than 10 IU/ml).
11. How can I maximise sensitivity?
As indicated above, the Company recommends an incubation time of 18 hours for maximum sensitivity (i.e., the ability to detect less than 1 IU per ml of sample).
12. What happens if I shorten the assay incubation period?
The assay becomes slightly less sensitive but remains quantitative at IFN levels between 10 and 200 IU per ml of sample. The following links show sample curves for 4 hour, 7 hour and 18 hour incubations respectively.
13. What happens if my samples are left to incubate for more than 18 hours?
Studies have indicated that assay variability is increased. The reason for this is not known; however, it may be due to the instability of the foreign protein (i.e., the firefly luciferase) in the cytoplasm of the human reporter cell. The longer the incubation, the more uncontrolled enzyme destruction.
14. Why is it important to use the test cells within 15 minutes of thawing?
The cells have been grown to a specific point in the growth cycle of the culture and treated to render them "division arrested". Cells that are thawed and allowed to metabolize may lose their IFN receptors and ability to respond to IFN in a way consistent with the performance claims of the iLite test.
15. What support does Neutekbio provide?
The Company provides complete customer service through phone or e-mail contact.
16. What quality systems are employed at Neutekbio?
Neutekbio follows the ISO 13485:2003 QUALITY MANAGEMENT SYSTEM. The primary objective of ISO 13485:2003 is to facilitate harmonized medical device regulatory standards for quality management systems.
17. Can the iLite Bioassay be used for diagnosis?
Yes. The iLite test has a CE Mark and can be used as an aid in the diagnosis of viral infections and to monitor IFN therapy in cases where the conventional IFN bioassay was previously used (i.e., France).
18. What is the batch size?
Neutekbio can supply batch sizes of 40 kits.
19. I intend to use the iLite kit to monitor IFN levels in clinical trials I am conducting. What is the batch to batch variability I can expect to see?
In a Performance Evaluation Study carried out with patient serum, the coefficient of variation in inter- and intra-lot analyses was approximately 15%.
20. Do I need to reserve kits from one batch for my studies?
No. Our Quality Management Systems guarantees repeatable and reproducible tests. However, we can send a single customer individual batches of 40 kits.
21. How has the assay been validated?
The iLite test has been validated through the completion of a Performance Evaluation as required for CE Mark (see Directive 98/79/EC and European Standard Document EN 13612). We have examined analytical specificity, sensitivity, accuracy, diagnostic specificity, reproducibility and repeatability. The study employed 300 patient samples.
22. What is the conversion factor for pico gram/ml or nano grams/ml to IU/ml?
If one assumes a specific activity for IFN of 2.6 x 108 IU per mg protein, then a picogram of IFN would be equivalent to 0.26 IU.
23. I have my own gene reporter assay and have no need to use one such as yours. What does yours offer that mine does not?
Ease of use, reliability, reproducibility and guaranteed performance.