While I have used GEL technology for many years, I have not used it to test Eluates. However, that has been the methodology where I recently started working. Just wondering who else out there tests eluates in GEL, and what problems you may have encountered?

We had one from last night that had a mixed-field "look" to it; but with cells streaming up through the GEL. I had another Tech. make a new Eluate and the testing came out the same. So I had the Tech. do Tube Testing with the Eluate and it was clean negative.

So.....

Thanks,

Brenda Hutson

We test our eluates in gel, the same way we do our antibody IDs. The only suggestion I can think of is that it's really important to get as much of the "junk" out of the eluate as possible before you pipette into the cards. It can cause a mixed field or weak to 1+ look if you accidentally suck some up, same as in the tube I'm sure.

I've done eluates in gel and I agree with Generic, the junk sometimes makes true reactions hard to read. And I think gel is sort of "super-sensitive" for lack of a better term. I'm not surprised that tube testing was negative.

I've always thought that an eluate was lots of science, a moderate amount of art and toss in some luck just to cover all the bases.

We have been running our eluates in gel for about 5 years. Rarely we do see the mixed field type reaction, however, for the majority of the tests the reactions look truly negative or truly positive.

I also run eluates in gel and occasionally have "junk" interference. If you transfer the eluate after the last spin in the eluate preparation and then pipette, I think the stroma at the bottom still gets mixed up a little. So I wait until after we pipette the gel card (drawing sample from the top of the eluate specimen), then we transfer the eluate to a clean tube. Seems to work for us.

Agreed, centrifuge the final eluate a couple times to make sure it is crystal clear, and you won't get that line at the top of the gel.

I agree. We have used gels for eluates now for almost a decade and, as long as the eluate is centrifuged "hard" for a short time to get rid of the "junk", we have had no problems whatsoever.

Edited March 17, 201213 yr by Malcolm Needs
Spelling - AGAIN!

I like that!

Brenda

I've done eluates in gel and I agree with Generic, the junk sometimes makes true reactions hard to read. And I think gel is sort of "super-sensitive" for lack of a better term. I'm not surprised that tube testing was negative.

I've always thought that an eluate was lots of science, a moderate amount of art and toss in some luck just to cover all the bases.

Thanks everyone! That is helpful.

Brenda

My lab plans to run eluate in gel, anyone care to share how they go about validating the test? Also, I am curious if anyone have experience running eluate using solid phase technology?

My lab plans to run eluate in gel, anyone care to share how they go about validating the test? Also, I am curious if anyone have experience running eluate using solid phase technology?

The way we validated the technique was to run the eluates in parallel in gel and by tube, in the same way that you would validate antisera. The experience was that they worked just fine, and we now use gel exclusively for our eluates.

We have not tried them in solid phase, as we do not use this technique at all in our laboratory, so I can't help there, but I would imagine that the principle is the same.

We run our eluates in Gel as well and aside from the problem with the junk we also make extra sure the pH is where it is supposed to be; we use an acid elution kit.