I am doing standard sandwich ELISA experiments and would like to know till what value of OD should be acceptable for the negative controls usually? I have used 7 rows for positive capture anti-bodies and one row for negative control. Then I use antigen and then a HRP labelled detector antibody for antigen.
The re should be least binding for the detector Ab in 8th row as no antigen would be present. There can be some non-specific binding which can give some positive signal. I am getting a signal of about 0.8-1 OD, while the positive capture Abs give a saturating signal of 4 OD. I would like to know how much negative control signal is acceptable? Is my control signal of 0.8-1 OD alright?
Hi Gupta,
Are you running a standard along with it? I assume you would be running one in every plate if you are performing a quantitative analysis. If you are, then the answer is quite simple, an OD value in the range of "zero standard" is acceptable. But, if you are running a qualitative assay, I would go ahead with analysis of the data considering there is a good signal to noise ratio (4.00 vs 1.00). However, I wouldn't recommend comparing samples between different plates as OD values for the same samples run on same parameters at different plate can be different. So, as far as you are running all your samples in a single plate, you can compare the data at this signal to noise ratio.
Hope it helps!
Hi Gupta,
Are you running a standard along with it? I assume you would be running one in every plate if you are performing a quantitative analysis. If you are, then the answer is quite simple, an OD value in the range of "zero standard" is acceptable. But, if you are running a qualitative assay, I would go ahead with analysis of the data considering there is a good signal to noise ratio (4.00 vs 1.00). However, I wouldn't recommend comparing samples between different plates as OD values for the same samples run on same parameters at different plate can be different. So, as far as you are running all your samples in a single plate, you can compare the data at this signal to noise ratio.
Hope it helps!
Even though your signal-to-noise ratio is not too bad, such high background levels are not normal at all. Are you sure your negative controls should be really negative? Usually background is lower than 0.1, sometimes is 0.1-0.2 or a bit higher, but in my case I dont accept a negative simple/control above 0.3 because this indicates that my ELISA is havig non-specificic backhground that can disturb my results. In such cases, changing dilutions and/or blocking/diluent conditions result in much lower background and highly positive signals.
OD of 0.8-1 in blank wells in abnormally high. As the positive control's OD is 4, the assay is working. You can reduce the OD values by increasing the wash steps, incubating in the wash buffer for 1-2 minutes at every wash step, change blocking solution, reduce primary, secondary, and substrate incubation times. Try incubating at room temperature.
An assay is acceptable if the Positive/Negative-Ratio is higher than 10. e.g. neg. control 0.1, positive control 1.0. According to lambert-Beers Law, the absorbance is the reciprocal values of the transmission. The absorbance is in a linear relation to the concentration. Sure, there are many instruments measuring absorbances up to 4.0. It’s due to the reciprocal relation nonsense. Try to bet neg. controls below 0.1, max absorbances not higher than 2.0.
You can regulate this by the reaction time for the substrate reaction (10 – 20 min are fine) or by the concentration (mostly not by the reaction time) of the conjugate (the labeled antibody).
Agree with the above answers. In general if negative control wells (average value) OD value is over 0.2, then I would not accept the assay, it could be because of the assay time, reagents, the plate itself etc.
Negative control values could vary from plate to plate and well to well. It is essential to have negative wells across different positions of any assay plate.
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