ELISA non specific binding - (Sep/06/2010 )

Hi!

In my lab we have an standardized indirect ELISA assay. Our antigens are produzed "in house" by cloning and expression in E.coli, followed by induction , extraction and purification (his-tagged) with ni-nta agarose.
After this purification , SDS-page and concentrations of these proteins are meansure.
After all, these recombinant polipeptides are used as antigen in indirect ELISa assay.
we coat the plate(Corning High Binding) with 4ug/ml of these proteins and incubate 48 h at 4ºC. After, wash twice with PBS tween-20 (0,1%) and blocked with 10% non-fatty dry milk in PBS tween-20 for 2 hours. Then 50 ul of the samples are added in dilution of 1:100 (the dilluent is 10% non fatty dry milk in PBS tween 20). incubate at 37ºC for one hour. After Wash three times with PBS Tween-20 and added secundary antibody with Peroxidase.Incubate for one hour at 37ºC. Wells are washed three times and then added ABTS. as positive control, Mouse immune ascitic fluid is used and anti mouse IgG is the secundary.
My problem is: Sometimes this works fine and well reproductible, but sometimes a high background in the plate even in negative control.

Someone told me to block with BSA fraction V, others told me to keep with milk.
Others told me to heat serum samples to inactivate complement (56ºC for 30 minutes), to avoid high background
How to avoid background?

What do you think ELISA's EXPERT about these tips? Any other tips for a begginer in Immunoassay.

Thank you

There are many, many blocking agents: milk, BSA, commercial blockers, etc. You can certainly do a screening test to see which one would be optimal for your work. make sure you completely fill the wells during the blocking step.

I believe you indicated you are screening ascites fluid? or ascites fluid is your negative control. Have you optimized the concentration of the conjugate you are using? Perhaps, the concentration is a bit too high and contributes to your background.

You may also add additional wash steps to reduce background.

Please describe exactly how much background signal you observe in your 'working' and 'problematic' systems.

just noticed...take the surfactant OUT of the blocking solution.

sgt4boston on Tue Sep 7 14:14:40 2010 said:

Hi
Thank you for replying me.

My ascitic fluid from mouse immunized with Ag against virus are my positive control and ascitic fluid from mice immunized with PBS is muy negative control.
I divided the plate in two: 40 wells for Recombinant protein purified and 40 wells for E. coli purified extract (null) as control of samples. Then i pippete samples in this wells in duplicate.
Sometimes high background is seen in negative control from samples (where I coated with purified E. coli extract)
My conjugate concentration is diluted 1:2000
this ELISa assay was standardized to screening human samples suspected of infection with Flavivirus like Rocio, Saint Louis Encephalitis and West Nile Virus.
These are the real problems faced: a high sensitive test, reliable, and with low porcentages of cross-reactivity ( as describle among Flavivirus family, low incidence of p-late background....

Would you say, to wash more times? Any other tips?

Thank you

I am a technical scientist at SurModics in Eden Prairie, MN. I specialize in our protein blocking/stabilization products. One major factor leading to inconsistent assay performance may be unreliable blocking.

We have 2 products which may help. StabilCoat (with bovine protein) and StabilGuard (completely synthetic). Both are great blockers which I routinely use in the lab, depending on the assay. Both can also be incorporated as your sample diluent.

If you would like to try a free sample (50 mL) for your next blocking buffer screen, please contact me at tjentz@surmodics.com.

More suggestions:

1. Instead of using the ascites fluid; do an ammonium sulfate cut and use the dialyzed product as a sample.
2. You can increase the number of washes and leave each wash in the wells for 1 minute before decanting and blotting.
3. Remove the surfactant from the blocking solution (make sure the wells are completely filled for blocking)
4. Surmodics, East Coast Biologicals, Candor Scientific, Kem-en-Tec each have proprietary blocking agents that you can examine...some will provide free samples...
5. Titer/dilute your conjugate further...

There is a challenge to having a sensitive assay with minimum backgroun