I'm a rookie in ELISA - (May/18/2010 )

Hi.

I want to start an ELISA protocol here in the lab, but right from the start. But neither I nor anyone in the lab ever did this. What I want to do is to measure the concentration of insulin on the culture medium of a cell line.

I have a primary antibody against insulin and a secondary antibody against the primary conjugated with peroxidase. What do I need besides this?

Thanks in advance.

-cardosopedro-

cardosopedro on May 18 2010, 12:49 PM said:

I think you'll need a block buffer and wash buffer, substrate for the peroxidase, plate reader, an automatic wash station for plates. Those are all I could remember. But I only did ELISA many years ago so my memory could be slightly off and/or outdated.

-ProteinWork-

Insulin measurement is done by 2 site sandwhich immunoassay. You need two Mabs; one for capture one for detection both of which are against different epitopes of the molecule. You may be able to use affinity purified polys or a combination of the two. The detection ab would be conjugated to some label...enzyme etc. You will also need insulin stock of know concentrations and a matrix such as serum, plasma etc devoid/stripped of the analyte and equivalent to the sample matrix you wish to test. Additionally, pre-analyzed samples run by a reference method which have values equally distributed over the analytical range of the reference assay (I am assuming your test will be equivalent). Let's not forget about plenty of time to do all the work.

It would be cheper and easier to just by a pre-existing elisa kit from one of the manufacturers and already on the market. If your samples are of different matrix than the standards they use you can make your own and use the rest of their kit components or use their kit and have a correction factor for any slope and intercept offset due to matrix differences.

hope this helps you.

-sgt4boston-

Hi,

I am working on an ELISA automation system where the entire ELISA process is automated. I want to find the dead volume by making use of the automated pipetting channels. What is the best method which I can follow?
Randomly put certain amount of liquid into the troughs and aspirate as much as possible? Somebody please give me an idea

-sagar3788-

Check out Aviva Systems Biology for ELISA Kits

-Kane for Aviva-

Here is the kit you are looking for:

http://www.calbiotech.com/store/product.as...=24&pid=101

http://www.alpco.com/products/Insulin_ELISA.asp

I belive the alpco kit is 510(k) approved so you can use those values for reporting and testing clinical samples.

-sgt4boston-

If you want to use a kit I like R and D system kits, they have an insulin one.

-labmeiser-