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.
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. …
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