In order to find the optimum concentration of my tracer, I replaced it in a commercial kit instead of kit's tracer, in the range of 5-150 ng/ml, but I was surprised to see the OD was similar and high in the whole range. (OD of kit = 1.9 , OD using my tracer in 5-150 ng/ml range = 1.9 – 2.1). Since the kit is designed to quantify analyte concentrations of up to 140 ng/ml, I think it’s not normal to get a maximum OD with labeled antigen as low as 5 ng/ml.
I would appreciate it very much if you could guide me if this is normal or am I doing something wrong?
Hi Yasman Moradi,
Can you please give us some information what you did?
So you did a competitive assay for a small molecule, it is a direct immunoassay, means the primary antibody is immobilized on the surface?, if I understood you right?
Now I am confused what you did:
1)You did a titration of your tracer in the range between 5-150 mg/L. and everywhere was the same signal? Then the answer is easy: You can even further dilute the Tracer, the OD shoudl be like 1 and when you dilute it further it should be less.
or
2) did you do a calibration curve (So you add your Competitor) in the 5-150 ng/mL range? with a fixed amount of Tracer? And you observe everywhere the same signal?
For case 2) This means, you need to dilute your tracer further more than that, you have too much tracer OR the Tracer binds non-specific to the plate. Did you block it?
I think, you might think the concentration of the Tracer should be the same as for the analyte? This is never the case, because they will have different affinities towards the antibody. Also you cannot do this in ng/mL, what "counts" here is the molar concentrations, because the Tracer, eg. a HRP Tracer has 40 000 Da and the analyte maybe 200 Da! So if you use the mass, you will get lost, because the affinity take the molar concentration into account (See "chemical equilibrium, The Equilibrium konstant, which is the affinity konstant, is calculated in mol/L)
The relationship between the tracer and the analyte is not 1:1 because the antibodies have different affinities to each the analyte and the tracer. You have to find out by titration, (Dilute the Tracer more) what is the best concentration. It depends as well on the incubation times. The shorter the time for the competitive reaction (e.g. 30 min is usually good) the better your sensitivity will be in the end. But for the moment use fixe values, to not making it more complicate.
A tip on titration
Do the Titration with Positive and negative controls. That means, one well you add the tracer in concentration X plus a blank sample (0 ng/mL analyte) and on the well next to it you use the same concentration of tracer plus a sample with 150 ng/mL of your analyte. In the first well the OD should be around 1 (adjust your Tracer concentration to this!) in the second well it should be almost 0 (like 0.002) You should include a negative control on that plate, were you add no Tracer. The OD of well 2 should be as close as positive to the negative control.
Best regards
Peter
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