When carrying out an ELISA immunoassay, there are certain doubts or questions that are frequently repeated when carrying out our experiment.
In this week’s entry, we bring you a compilation of frequently asked questions about ELISA kits prepared by Biomatik , which can help you answer some questions.
1.- HOW SHOULD ELISA KITS BE STORED?
Although certain kits may have specific storage conditions that will be described in each case, in general, the components of the ELISA kits can be stored refrigerated (4ºC) or frozen (-20ºC).
2.- HOW DO I PREPARE THE REAGENTS?
The reagents must be prepared 10 minutes before use. When first used, reagents should be concentrated by centrifugation at the bottom of the tube.
Frequently, the amount of reagent in the tube is usually greater than the specified, so it is recommended to measure the amounts to be added accurately with a pipette or similar.
3.- HOW DO I SEPARATE MY ELISA PLATE?
The well strips of the plates are mobile, so it is recommended to store those strips that are not going to be used immediately between 2-8ºC and in dark conditions.
4.- HOW DO I ADD THE SAMPLE AND / OR THE REAGENTS?
All samples should be added over a 5 minute period. The time between each sample addition should be uniform to ensure consistency of the reaction.
5.- HOW DO I INCUBATE THE PLATES?
Proper use of new, clean plate sealers must be ensured to avoid sample evaporation and contamination. When moving the plate, you must keep your hand steady so as not to spill the liquid. In order to maintain a constant temperature of 37ºC, excessive opening of the incubator door should be avoided.
6.- HOW DO I WASH THE WELLS?
The same volume of buffer should be used per well, ideally with a multichannel pipette. When using an ELISA plate washer, it must be ensured that it is clean and free from contamination. The plates should be gently tapped face down on the filter to dry them properly.
7.- HOW DO I READ MY ELISA PLATE?
In order to avoid reading errors due to accumulation of precipitate, the plate should be read within 5 minutes of adding the stop solution. Make sure that the microplate reader is properly configured and the filter is properly calibrated. On the other hand, avoid mixing reagents with different lot numbers.
8.- CAN THE STANDARD CURVE BE EXTRAPOLATED?
Out-of-standard curve results are not supported. Only those results that fall within the standard curve are reproducible and therefore accurate, according to the kit.
9.- WHY SHOULD I DILUTE MY SAMPLE?
If the values are above the standard curve, the samples should be diluted to fit the detection range of the kit.
10.- WHY DO I GET LOW SENSITIVITY AND / OR ABSORBANCE VALUES?
You need to make sure that the target protein is expressed in your sample. In case the expression level is low, an attempt will be made to increase the amount of sample used. In some cases it may be necessary to resort to a higher sensitivity assay. It is also important to confirm that the positive control being used is within the detection range of the assay.