ELISA Testing and results
A: There are 3 conditions for interpretation of test results:
1. The test results must be valid and accurate. This can be done by running a sample with a known value together with the new (your) samples. BioChek laboratories usually do so. The name of the known sample has the prefix RF or RS. Please check with the laboratory
2. A statistically valid number of samples should be tested ( interpretation manual 2012 vs 3 page 6)
3. One should know what result to expect prior to sending out samples for testing (interpretation manual page 18 and further)
A: This means something went wrong when running the test. Please make sure you’re using the correct range corresponding to the reference control sample used. Contact BioChek for more help
Q: What should I do when a low number of samples test positive when expecting 100% negative results?
A: Re-run the positive samples. Make a fresh dilution and test again. If still positive, use an alternative test for confirmation. Only run the positive samples!
A: No, not for ELISA purposes. (The term serum relates to blood plasma without clotting factors like fibrinogen).
A: The kit controls only show the kit differentiates between positive and negative samples. The reference control will confirm quantitative accuracy. Think about it like this: a straight line is defined by two points, however to fix the angle of the line one needs a third point.
A: In qPCR, the amplified DNA is labelled – usually with fluorescent dyes enabling amplification of DNA visually by reading the fluorescence of each well. Fluorescence is measure after each cycling enabling testing to be read/analysed in real-time. Conventional PCR can only be visualised by running amplified DNA samples thru electrophoresis. This is done at the end of the cycling run. Conventional PCR can also be referred to end-point PCR. In qPCR, the amount of the fluorescence released during amplification is directly proportional to the amount of amplified DNA. There is no electrophoresis step needed to visualise DNA for qPCR, but is required for conventional PCR. The steps for annealing and extension during replication of DNA is usually combined during qPCR testing.
A: In-house kits are not as rigorously quality checked as commercial kits and can vary from batch to batch. Most are not validated with as many different species as a commercial kit leaving them more vulnerable to cross-react. Most are not checked periodically with new species/strains which may lead to false negative results.
A: Antibody detection is best used to determine the optimal moment for vaccination and to confirm correct timing and application of the vaccine.
A: This indicated the lowest limit of detection for our kit. For example, we can detect any Salmonella positive sample down to 100 copies of DNA which below infection rates.
A: Yes you can. All our kits have the same protocol on the thermocycler so you may run our kits at the same time. The only stipulation is if you run a RNA kit alongside any of our DNA kits, you must always run the RNA thermocycler protocol. The RNA protocol has an extra heating step to make cDNA.
A: Yes, you can. When using the standards we provide, you can calculate relative load.
A: You need to use standards to determine quantification of positive samples.
A: Please use them as you would a sample or a control. You will take 5ul of each dilution into the PCR A: reaction mix with the same procedure and conditions.
A: Yes, BioChek software has a PCR function on it. Demonstrations can be done to show how it works or a manual can be given.
There are many thermocyclers currently on the market that are suitable dependent on the specific PCR kit. Any thermocycler that uses the correct dyes for detection that is required for our kits will work. The dyes are important and not the manufacturer of the thermocycler. Currently the most commonly found compatible PCR cyclers for all BioChek kits are as follows:
- ABI 7500 or 7500 FAST
- BioRad CFX 96
- Agilent Mx3000 / Mx3005
- Roche Lightcycler 480
- Roche LC96
- Qiagen Rotorgene Q
- Corbett RG-6000
- Thermofisher QuantStudio 5
Other thermocyclers will work if the filters used are correct for the specific test kit used. If a dye is missing from the machine that is used on the kit, the kit may still be used, but the customer will sacrifice a target being detected. However, the internal control cannot be sacrificed. You will not be able to use the kit if the machine does not detect the specific dye used for the internal control.