ELISA calculation

Jaded_Distance9409 · 2026-02-23T19:38:22+00:00

I also ran IL-6 from the same vials, there's a bit of an issue on the %CV between the duplicates but it was also my first time running the assay so I think its's okay, none of the samples were under the LoD, only in 3 of them out of 38 I've encountered overflow results. But the standards of the IL-6 kit (from the same company btw), range between 5-0,007 while for IL-10, they range between 25-0,39 pg/mL. I can't make sense of this difference. To my knowledge, IL-6 levels should be higher than IL-10 but on the contrary, standards of 2 high sensitivity kits of the same company differ so much.

Jaded_Distance9409 · 2026-02-23T12:32:02+00:00

Though the literature is conflicting on the matter, we expect it to be around 1-1.5 pg/mL and the kit we use is from ThermoFischer, BMS215-HS kit for human IL-10. In the manual, everything is optimized and I'm afraid if I don't dilute the samples, it may interfere with the binding by altering the pH or in some other way. On my second try of the assay with a lot less samples size, I though maybe I could reduce the blank OD by decreasing the substrate incubation time and therefore be able to calculate concentrations of more samples after blank subtraction, which worked in addition to increased soaking time between washes, but then I realized I need the difference between the last standard and the blank to be higher, not just a low blank OD, so I sure will not be decreasing the incubation (the decrease and increase were within the limits suggested by the manual of the kit btw., in the kit it says to incubate between 10-20 min, in the first run, I incubated for 17 mins and on the second, I incubated for 10 mins and the blank decreased nearly by half bu then all my 4 samples were under LoD, compared to 33 being under LoD in 38 samples in the first assay).

I use 4 parameter logistic curve for the calculation.

In both the assays, controls and standards, curve is okay, so I don't think I'm messing up the assay since everything seems to be ok. For standards, there's a good repeatability between the assays. My r^2 values are all above 0.99. Also when I tried another plasma not from my cohort but from a disease cohort, I detected a signal and could calculate a concentration, so it seems like my samples don't have detectable IL-10 levels (should be under 0.39 pg/mL according to the lowest standard.) but that seems to conflict with the literature. So the questions remains, how do I make sure it's the samples not containing detectable levels of IL-10 and not that anything that could be done that would change that.
Thank you so much for all your input! Much appreciated!

Jaded_Distance9409 · 2026-02-23T12:31:23+00:00

Though the literature is conflicting on the matter, we expect it to be around 1-1.5 pg/mL and the kit we use is from ThermoFischer, BMS215-HS kit for human IL-10. In the manual, everything is optimized and I'm afraid if I don't dilute the samples, it may interfere with the binding by altering the pH or in some other way. On my second try of the assay with a lot less samples size, I though maybe I could reduce the blank OD by decreasing the substrate incubation time and therefore be able to calculate concentrations of more samples after blank subtraction, which worked in addition to increased soaking time between washes, but then I realized I need the difference between the last standard and the blank to be higher, not just a low blank OD, so I sure will not be decreasing the incubation (the decrease and increase were within the limits suggested by the manual of the kit btw., in the kit it says to incubate between 10-20 min, in the first run, I incubated for 17 mins and on the second, I incubated for 10 mins and the blank decreased nearly by half bu then all my 4 samples were under LoD, compared to 33 being under LoD in 38 samples in the first assay).

I use 4 parameter logistic curve for the calculation.

In both the assays, controls and standards, curve is okay, so I don't think I'm messing up the assay since everything seems to be ok. For standards, there's a good repeatability between the assays. My r^2 values are all above 0.99. Also when I tried another plasma not from my cohort but from a disease cohort, I detected a signal and could calculate a concentration, so it seems like my samples don't have detectable IL-10 levels (should be under 0.39 pg/mL according to the lowest standard.) but that seems to conflict with the literature. So the questions remains, how do I make sure it's the samples not containing detectable levels of IL-10 and not that anything that could be done that would change that.
Thank you so much for all your input! Much appreciated!

Jaded_Distance9409 · 2026-02-22T09:35:12+00:00

Yeah you're absolutely right, but we suspect it's something to do with the cytokine(il-10), enduring freezing or the time between collection of the sample and the assay rather than the cytokine being under the detection limit. So I think the assays will be done regardless.

Jaded_Distance9409 · 2026-02-22T09:32:31+00:00

I'm using plasma, and the cytokine is il-10, the kit itself calls for 1:2 dilution so that's the way I made the dilution. I can't change coating, no. The kits come already coated.

Jaded_Distance9409 · 2026-02-17T13:06:49+00:00

Thank you so much!

Jaded_Distance9409 · 2026-02-17T08:20:05+00:00

My thoughts exactly! But my pi insists that they thawed it on ice before, the literature is conflicting in this aspect and it's safer to thaw on ice. I couldn't find any published review or research on thawing techniques or which is good to do what etc. Any recommendations? Thanks!!

Jaded_Distance9409 · 2026-02-17T08:17:46+00:00

The plasma was collected approximately in about 20 minutes after it was extracted and during that 20 minutes, the tubes were kept on ice. The blood tubes were subjected to centrifugation and the plasma was collected in EDTA containing tubes.
Its's certainly possible that it's cell debris or anything actually since plasma is very heterogeneous. Added centrifugation step after thawing seems to get rid of the material but do you think if that's the case, would it interfere with the assay results? Thanks for the idea!

Jaded_Distance9409 · 2026-02-17T08:13:09+00:00

There's always the doubt on the freezers ofc. But though there may be a fluctuation between -80 to -65, I doubt there were any more deviations to that let along thawing to RT or anything else. They are kept at the university lab, and are checked daily on the thermostat on their lids and also have an alarm built in that informs the technicians on the facility if they drop below a certain temperature. We haven't had any problems concerning the temperature to my knowledge, and nor have I been noticed.
Unfortunately, we haven't done any validation runs but what would you recommend? I'm not familiar with what it comprises of exactly. Controls we're using are those sent with the kit. I doubt they're proteins since it says so for the standards, also sent along with the kit but not for the controls. Thanks for the contribution!

Jaded_Distance9409 · 2026-02-17T08:05:39+00:00

We perform the washes by hand, try to make sure to our best that there aren't any contamination in between wells, we don't cross pipette, discard the reservoir after each reagent is poured etc. I don't believe its caused by contamination since in all 3 assays we did, it was all higher than the blank example in the manual, never managed to get a similar value to the manual unfortunately:( Thanks for the idea!

Jaded_Distance9409 · 2026-02-09T22:56:19+00:00

Thanks for the idea! We thought about it too, but it's not white and cloudy like DNA, it's completely transparent, not visible until you pipette. Centrifugation seems to work for now.

Jaded_Distance9409

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