Protocol Online logo
Top : New Forum Archives (2009-): : ELISA and Immunoassay

ELISA plate layout - any recommendation? (Jul/08/2009 )

Hi, i going to run my very first ELISA using commercial kit. However, since this is my "virgin" kit ^^, just want to know what plate layout you would suggest from your experience? I need to run abt 25-27 different samples on a plate and since it is very expensive, i believe choosing a right plate layout can help to avoid unecessary waste of well. what would you suggest, i.e duplicate standards/duplicate or triplicate samples/positive or negative controls? btw, i just got to know that if the serum/plasma concentration is too high, we may need to dilute before we run, but how we measure its concentration?



One advice, follow the instructions in the kit very carefully esspecially the washing instructions.
The kit will mostly give also a plate setup and always every in duplo, standards controls and samples.
The dilution you need will also be recommended in the kit, whenever I use a kit for the first time I almost always follows the given instructions without any change.


Follow the instructions. If you dilute a sample just mutiply the result by the dilution factor. Do you know if you may have samples "below" the lowest postive standard/calibrator? Sometimes I incorporate a point between the 0 and the next standard and/or also run the highest one at twice the volume for a higher calibration point.


Thanks! i got another question: what is the blank, positive control and negative control? Lets say i got some plasma samples which are slightly hemolyzed, the plasma appeared slight red, should i prepare blank for this? just want to assure this so i won't make bigger mistake that can waste my precious wells.



The manufacturer can clarify or should indicate in the package insert if hemolyzed or lipemic samples can be tested. Positive and negative controls should be material of the same "matrix" as samples that can be analyzed by the kit. Usually, the manufacturer supplies controls or they are available from another source. Controls should come with information as to the anticipated value the control should be. If outside the range then run is rejected (along with other parameters you should be monitoring such as curve slope, %CV of replicates, fit of the curve, midpoint, 20 and 50% binding points...i know this is quite a bit of info but at least you know what to be looking for). They run controls multiple times to establish the range. Kit manufactuers most often supply controls which are nothing more than extra standards. Use controls from another source if possible OR if you have access to previously run samples those could serve as your control (but you will have to allow some variation ie +/- 5-10%).



In fact, i wonder i shd use duplicate or triplicate? the reason why i use duplicate is because i want to save wells, especially when some samples are out of ranged and need dilution and re-do. Therefore, i thought i can run all my samples in duplicate first, and when one of my samples, the variation is too wide i still can do another extra well next time (to replace the one with high variation value). Do you think i can make it this way? or does the result actually can be different even using same samples, but analyzed in different time? Thanks


I have started to do elisa recently but the most i found useful was to do the elisa standards in triplicated of 12point serial dilution with a start concentration of 1ug/ml , and do the samples in duplicates, and last row use for the blank
This will help you to know the conentration of your standards are of the right concentration which would be easy to analyse the samples.
I hope this make sence :)


If your technique is good and kit is expensive run duplicates for the standards and singletons for the samples. If you have an in-house kit there is no need to run more than 8 points (one column) on your plate. If you need to cover a wide range use serial 1:3 dilutions. Be sure to run samples reading greater than your highest std at a higher dilution using the diluent supplied in the kit (if no diluent use the 0 std or lastly a PBS/BSA (without surfactant)

Good luck!