Why analysis protein with ELISA, often have wide range standard deviation?
An ELISA has normally if exactly carried out, an intraserial imprecision below 4% and an interserial imprecision below 6%. The reasons for higher values are quite different:
Mostly found:
1. Temperature gradients during incubation (overcome by temperature equilibration before the assay starts, no incubation at 37 °C or at 4 °C., temperature gradients during coating)
2. Humidity gradients during coating and incubation (overcome: cover the plates)
3. Solid phase contacts during pipetting which destroys the solid phase coat
4. Inconstant incubation times = time gradients over the plate caused by slowly pipetting (overcome: use multichannel pipettes and prepare the samples in a separate preparation plates which allows the sample transfer for the whole plate within a minute or use longer incubation times: example 15 min pipetting time for 60 min incubation time 25% variance, 16% in 90 min incubation time …)
5. Inconstant incubation conditions due to different sample protein content, different sample pH, … (overcome: use an optimal dilution which should be higher than 1:10, best 1:50 and an buffer which allow the constant conditions: 0.01 mol/L phosphate pH 7.2, 0.3 mol/L NaCl, 3% v/v Gelafusal [digested gelatin with normally no cross reactions etc], 0.1% v/v tween 20, 0.001 g/L phenol red.)
6. Inconstant incubation times for the second incubation
7. Inconstant incubation times for the substrate & stop solution
8. Pipetting of bubbles in the wells
9. …
Maintaining the uniform and method given conditions (viz. temperature) and uniform liquid dispensing (pipetting) during the steps of assay procedure can help in reducing the STDEV as well as %CV.
Storage conditions of your controls also matters there.
I mean all the analyte specific conditions (to not to denature your protein) and assay plate too.
I believe that the amplification that occurs in the assay makes it very sensitive to small variations in technique, as discussed by Lakshmi.
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