Make your own ELISA! We all used to do it before kits, which are expensive. Source a good primary anti-insulin antibody with which to coat the plates and an appropriate PO-conjugated antibody for visualization. Much cheaper than kits, and really quite straightforward with appropriate standardization and controls.

Thanks for your answer! I was looking at trying to find a 'make your own ELISA kit' but couldn't find one...guess I might have to go the 'old fashioned way'. Though I don't have much experience with it...do you know a good reference I could look at which contains protocols for on coating my own plates and developing appropriate standardization and controls? Also, do you have any suggestions for a few companies that might supply good antibodies for insulin and visualization?

You could have a look at this ResearchGate discussion as a good starting point:

https://www.researchgate.net/post/Making_our_own_ELISA_plates

Also LifeTechnologies have quite a lot of resources (although probably not the cheapest!). In the UK this link works - not sure in Canada - you might need to search "Buld your own immunoassay" on their website:

http://www.lifetechnologies.com/uk/en/home/life-science/cell-analysis/immunoassays/build-your-own-immunoassay.html

Is this for human insulin in plasma?

You need to be careful because there is poor comparability and standardisation between kits. The limit of detection can also be an issue. Mercodia makes a good ELISA that is used in some of the clinical labs in the UK.

Thank you for your answers. I will have a closer look at the websites suggested...a former colleague mentioned it would be easier if there was already a 'make your own insulin ELISA protocol' as it would give me a starting point with incubation times etc. Is this the case? If so, does anyone know any labs that make their own human insulin ELISA plates that might be able to help us out?

Try Mercodia Elisa Iso Insulin kit, which has a good price. If you don't have any experience with home making ELISA and your samples are valuable do try it. Is there a possibility to use electrochemiluminesence (Roche instrument). The price per sample is relatively low

Good luck

Hi Sylvia,

How does HTRF price per sample compare to EIA? I don't have experience with it, but it is said to be very sensitive. I usually use RIA unless sample volume is an issue. I use the "Linco" Millipore EIA and RIA, because they compare well to each other. I am in the process of testing the new Stellux chemiluminescent assay, which works well, but I have not tried it side by side with my usual kit yet. I have has good results with Mercodia, Crystal Chemical, Alpco, they all work well. Ask your sales rep for a sample and try them side by side. Try HTRF as well if you have a plate reader that can do that. Mine doesn't.

If you want to design you own EIA, divide your 96 well plate in half, one side is for your positive, the other for negative. Coat the plate with anti insulin, titered across the positive (wells A1-H6) and negative (wells A7-H12) sides of the plate. You will put 50 ul coating buffer per well except for Column 1 and 7. In 1 and 7, you will put 100 ul of your most concentrated anti insulin in coating buffer. Titer using an 8 channel pipette that can be set to 50 ul. When I used to do this, I used silanized glassware to prepare the coating buffer, and prepared it immediately before use, because you have no other proteins in the buffer and even with silanized glassware, you may lose some antibody to the glassware.

Wrap the plate in plastic wrap and leave overnight at room temperature. Wash with PBS/Tween and block with PBS/tween with 0.5-1% BSA added.

On the left side, leave A1 as your blank (do not add conjugate to A1). Prepare a solution of insulin that represents the highest insulin concentration the assay will measure. 10 ng/ml is a reasonable concentration. Prepare negative control solution, which would likely be your assay buffer. Add 5-10 ul/ well of the positive to the left side, and negative to the right. Allow to incubate for two hours, maybe longer if you are going to try to minimize antibody requirements.

Wash plate. This time, you will titer the secondary antibody down from row 1, making sure to omit conjugate in A1. The rows B-H will have 100 ul of assay buffer. A2-A12 will have 200 ul of your most concentrated antibody conjugate. 1:100 is a reasonable starting point unless you are purchasing conjugate and can get manufacturer recommendations. Titer the conjugate down the plate. Incubate for 30 minutes. wash well. Apply 200 ul substrate (TMB or ABTS are good). Incubate until sufficient color development occurs, which could be from 5 to 30 minutes. Read on the appropriate wavelength.

You are looking for the plate coat concentration and conjugate concentration that give you maximum signal at 10 ng/ml insulin, with minimal signal in the negative control at the same conditions.

This is easy enough to do, but when you are calculating your savings, remember that you will want to do adequate QC on your system each time conditions change. If you are making your own plates, you may not know the shelf life of the plates if you make a batch and dry them down for future use. If you make your own antibody conjugate, there will be some variability there as well. One thing you pay for when you buy a commercial kit is the QC and minimized variability.

Alternatively, is there a core facility that does assays that you might access? For example, I get special pricing at University of Washington, so many samples are cheaper for mw to send out than to run in house. CR-CHUM, maybe?

Another possibility is that you might be able to eeduce sample number. Begin by running complete sets, but look to see if there are samples that do not give valuable information, so that maybe you can reduce the number of samples you need to assay. We do perifusions and in new experiments, we tend to test every minute, but after a few, we assess which samples are really necessary so we don't end up running 1000 tube RIAs. 600 tube, maybe, but not 1000!

Good luck!

Elizabeth

ELISA method is cheap and accurate.

according to http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3290000/ 

silanized glassware greatly reduces insulin recovery!

polypropylene should be avoided too.