I am trying to develop an ELISA to measure Ab against aden associated virus (AAV). I am using Nunc-Immuno™ MicroWell™ 96 well solid plates (Sigma-Aldrich; Cat.# M9410-1CS) to coat AAV using different conc. of PBS (0.5X, 0.2X and 0.1X). Coating is overnight at 4 degree followed by washing 3 times with 1X wash buffer (10X wash solution (from ImmunoChemistry Technologies). I tried blocking the plates with different blocking buffers (5%BSA, 2% Milk and 5% milk), washed 3 times and proceeded with serum and Ab incubation. All incubations have been performed at 37 degree for 1 hr followed by 3 times washing. I used different buffers to dilute serum and antibodies (0.1% BSA, 0.5XPBS, 0.2X PBS and 0.1X PBS). Secondly Ab is anti-human IgG HRP from ThermoFisher. I am getting a high background throughout the plate and need your suggestions to solve the issue.
Thank you so much for suggestions.
Here are few things you could try, but first two questions: How clean is your antigen? Have you tried to detect your antigen with a commercial ELISA?
Remeber: never change more than one thing at a time!
1. Use a buffer with detergent, eg. TWEEN-20 (PBST). Try 0.1% of TWEEN20.
2. Try shortening the incubation times @37C to 30 minutes.
3. Coating the antigen on a plate may be a tricky Thing. Would you have a possibility of coating first with a commercial monoclonal Ab against your Antigen? You will lose one epitope but probably gain.
4. What do you use to develop? Did you check if your reagents are still good?
Agree with Agnieszka.
Also try the following:
1. A dilution range of the serum. Your current primary antibody concentration may be too high.
2. Are you using recombinant virus protein for coating the ELISA plate? If not, what is the source of the virus. If it is isolated from human tissue/cells, you may have contaminants causing high backgrounds.
3. Add some of the anti-human IgG-HRP to ELISA substrate in a small tube and check if you see a colour change. This will tell you if your detection system is working.
Another option: how do you wash? I found immersing the plate in wash buffer for 4-6 times and vigourously shaking out any buffer really helps with background.
Have you tried to perform incubation at room temperature instead of 37°?
1. Use blocking solution (for instance PBS+4% milk) not only to block the plates, but also to dilute samples and conjugate...
2. Try to dilute moere your conjugate to find a goosignal/noise ratio.
Thanks all for your suggestion.
@ Agnieszka I am planning to try both detergent as you mentioned and short incubation time @ Edmund virus is produced from HEK cells and is purified using CsCl gradients followed by saline buffer exchange. Detection system is working as I do see color in PBS only control.@ Victoria I use hand wash using multi-channel pipette. @ Gertrudis I am planning to use 1XPBS, 2% BSA, .1% tween 20 as incubation buffer.
What happens if you do not add your primary antibodies and only add the antigen and conjugate. If it is high with just the conjugate first dilute the conjugate more. Second step is to look at the antigen. In what media is the virus grown. You could have IgG from that specie (fetal calf serum) in your assay that the detection could react with. If so, you could desorb the antigen with protein A or protein G sepharose. You could try adding 1ul of whole serum from the same specie. This should reduce any reactivity with IgG in the coating antigen. Use tween in the incubation steps and possibly increase the number of washes (from 3 to 4-6). Empty the wells carefully by hitting the emptied plates against a stack of tissue paperafter each washing cycle.
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