What Can I Do to Get Greater Q-Plex Sensitivity?
If your antigen concentrations are very low, we first recommend that you select a High Sensitivity or Singleplex Q-Plex™ Array. The assays in these kits have specifically been optimized to have the greatest sensitivity possible.
If you already have a normal kit, you can try the slightly modified protocol and data analysis tips below. Consider changes to the assay protocol carefully, however, as this can also have undesirable side effects.
Modified Assay Protocol for Increased Sensitivity
(Use at your own risk)
To optimize for detection at the low end of the curve, extend the standard curve a few extra points, and use longer incubation times, for example:
|Normal Human Cytokine Protocol||Example Modified Protocol|
|Calibrator Curves||Start Neat, do 1:3 dilutions out 7 points||Start Neat, do 1:3 dilutions for 5 points, then 1:2 dilutions for 6 points, for 11 points total|
|Incubation Times||Antigen: 1 hour Detection: 1 hour Conjugate: 15 min||Antigen: 2 hours Detection: 2 hours Conjugate: 20 min|
This modified protocol sacrifices some quantitation (1-2 points) at the top end. Also, some samples may have increased background with longer incubations, which will decrease sensitivity.
Data Analysis Tips
To optimize the curve fit for the low end, first, mask standard curve points if their % backfit is not 80%-120% (shown in the Report tab of Data Analysis). Next, mask the top 2-3 points of the standard curve if they haven’t already been masked. Finally, choose Auto-Select the regression model and auto select favor low for weighting. This may provide a better fit at the low end of the curve.
Can I increase incubation temperatures?
This is not recommended. We find that increasing incubation temperatures can cause antigens to degrade and non-specific binding to increase.
Can I run my samples undiluted? Or less dilute?
This is not recommended for most sample types. Cell culture media is the only sample type that can be tested neat, but all other sample types should be diluted at least 1:2 (50%) for use in Q-Plex Arrays. This is because of the increased likelihood of getting false positives and high variability between replicates with neat samples – rheumatoid factors and heterophilic antibodies such as human anti-mouse antibodies are usually the culprit in blood-derived samples, while high total protein or lipid content often causes well background problems with tissue-derived samples. Q-Plex Array diluents have additives to address these issues. If you are concerned that false positive signals may occur in your samples, test them at several dilutions, such as 1:2, 1:20, and 1:200, and check that the dose-response is linear. Signal from heterophilic antibodies will often not dilute out, while real signal should get dimmer as the sample is diluted.