Hi researchers,
Is it necessary to have a negative control for ELISA? I see someone mentions that negative control is serum without my aim protein. If it is so, how can I get such kind of serum?
Another question is about the quality control of the serum. Because I hand over the samples from others, if I want to check the quality of the serum, what should I do? I mean things like hemolysis. Or if the serum preparation is proper, there is no need to check the serum quality?
Thank you for your reply!
I cannot think of many cases where you would NOT want a negative control for ELISA, so I think it is pretty necessary, much as a positive control is necessary. About the time you don't use a negative control is exactly when every sample you test will be positive and then you'll really be left doubting the results. As far as negative control sera, that's a matter of finding an animal/patient lacking the antibodies that you're testing for. There's really no other way around it.
In terms of quality of sera, most of the problems you'll see are grossly observable, such as hemolysis (pink to red), lipemic (looks milky or cloudy), and icteric (looks very yellow, almost like urine). In some assays, sera with these conditions will still work but it needs to be tested.
I cannot think of many cases where you would NOT want a negative control for ELISA, so I think it is pretty necessary, much as a positive control is necessary. About the time you don't use a negative control is exactly when every sample you test will be positive and then you'll really be left doubting the results. As far as negative control sera, that's a matter of finding an animal/patient lacking the antibodies that you're testing for. There's really no other way around it.
In terms of quality of sera, most of the problems you'll see are grossly observable, such as hemolysis (pink to red), lipemic (looks milky or cloudy), and icteric (looks very yellow, almost like urine). In some assays, sera with these conditions will still work but it needs to be tested.
Negative control serum could be obtained from "knock out mice" if the serum sample is from mice. Another way is to deplete your protein of interest by IP using a validated antibody and confirm by western blot that your protein of interest is depleted completely.
Regarding serum quality, Josh answered it.
Hi Josh,
Thanks for your reply. Now I have positive control, it's the pure protein I want to examine in serum. But for negative control, I really don't know how to get the serum without the protein. It's supposed to be in both patient and healthy people.
If use animal serum, how to get it? And in this case, how can confirm the protein in animal won't interact with my antibody?
I do see some cloudy samples. Thanks for your explanation. It's really useful!
I only said animal serum because I work in the animal health industry so I'm making assays for animals. You may have to try depleting the target protein as Mike has mentioned, however I do not know much about that technique.
Actually, don't you have a standard curve for your analyte on the plate? And, is the ELISA homemade or has it some certification/validation for the intended use?
Depleted serum would be the ideal control, as already mentioned. To prepare some, immobilize antibodies against your target protein on reactive resin (NHS activated sepharose etc) and pass a serum sample across it. If necessary several times (usually, the affinity column may be regenerated by a wash with glycine HCl pH 2.5 or similar).
In order to confirm that the ELISA system itself is working and not producing false positives, analyzing a sample of sample diluent oder BSA solution in PBS, Ringer or similar might do the trick. Also spiking serum samples with known amounts of target protein and checking the recovery rate should be suitable to prove that your test is working.
Hi Mike,
Thanks for your reply. I don't think I can use "knock off mice" to generate the serum.
And I wonder whether every ELISA has to do the negative control? I mean in some publications, they didn't use negative control.
Hi Wolfgang,
The ELISA plate is a commercial one and there are 6 diluted standards to make standard curve.
Do you do negative control every time? I mean if I make the negative control from generation antibody, ELISA is such a big experiment.
Did you mean I can use BSA instead?
Thanks!
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