unreproducible results

[Marshall]

Hi All,

I'm trying to measure serum IgG levels in bovine calves using the "Bovine IgG Quantitation Kit" from Bethyl Labs. My standard curves are consistently reproducible and have very high r-squared values. I run each sample in triplicate and the values of each replicate are generally quite close (w/i 10% or better).

However, when I run the same serum samples on different days I often get very different values. Not only are the values different, but there is no pattern to the differences - for example, sample 1 might be higher than sample 2 in one assay, and the opposite case in the next. Sometimes there are wildly different results, an order of magnitude or more. It's as if the serum was non-homogeneous, despite my sincere attempts at mixing (inversion and vortexing).

A few notes: the samples are collected in Corvac serum-separator tubes, centrifuged at 3220 x g, the serum transferred to cryovials and frozen at -20 until the time of assay. The samples I have re-tested were refrozen and rethawed. In order to get the samples in the "linear range" of the assay, I am required to do a 1:800,000 dilution. However, the reproducible standard curve is a dilution range of 1:48,000 to 1:3,072,000.

Any ideas?

[danfive]

Freeze-thawing is your first and worst problem.
Try:
1) aliquoting blood samples, use once--don't re-freeze and thaw.
2) adding proteinase inhibitors, sodium-orthovanadate.

[Zagami]

The human serum (off-the clot), but bovine serum anymore, to form cryo-aggregate when stored at -20 °C. This phenomenon verified during the frozen, but also in thawed step. In addition, when the sample serum is refrozen and rethawed more times, beyond the deterioration of the molecule to be examined, the cryo-aggregation increases. Such presence cannot be eliminated with some mixing, even if "sincerely attempts" but through the use of organic solvents. I allow me to offer you an idea : layer on 1 ml of bovine serum 1 ml of ethyl ether and, without absolute mixing of the two liquids, incubate at 4 °C for 30 minutes (optional). After, centrifuge at 1500 rpm for 5 minutes or more, in turbidity relationship, discards the supernatant solvent, helping with a pipette Pasteur connected with a vacuum water pomp, up to some over the interface with the serum and therefore employ serum for the test.
Note : for the experimental conditions, re-examination, induces to believe that a molecular denaturation is verified with consequent evidence of different epitope by those wanted from the first antibody at the solid surface coated. In more, conformational modifications can mime researchable epitope, that can explain the differences between first and next sample processing. As says again, cryo-lipidic-aggregate (CLA) can adhere to the surface of the solid phase making it insensitive to the second antibody, or to exalt its avidity. At 1:800,000 dilution (very high in consideration that in human serum concentration of IgG is ranged from 4 to 16 mg/ml and sensitivity of microtiter EIA is 1x10-9 g/ml), it shows that to this dilution the "critical factors" don't modify the specificity.