Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > Protein Science > ELISA Assay Forum
Register Search Today's Posts Mark Forums Read

ELISA Assay Forum ELISA Forum. Discuss and post questions including optimization, troubleshooting, protocols and theory of ELISA assays using monoclonal, and polyclonal antibodies or peptides.


unreproducible results

unreproducible results - ELISA Assay Forum

unreproducible results - ELISA Forum. Discuss and post questions including optimization, troubleshooting, protocols and theory of ELISA assays using monoclonal, and polyclonal antibodies or peptides.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 08-18-2009, 06:34 PM
Pipette Filler
Points: 46, Level: 1 Points: 46, Level: 1 Points: 46, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Aug 2009
Posts: 2
Thanks: 0
Thanked 1 Time in 1 Post
Default unreproducible results



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?
Reply With Quote
  #2  
Old 08-18-2009, 07:38 PM
danfive's Avatar
Nobel Laureate
Points: 8,612, Level: 64 Points: 8,612, Level: 64 Points: 8,612, Level: 64
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jul 2007
Location: Houston TX
Posts: 969
Thanks: 12
Thanked 184 Times in 154 Posts
Default Re: unreproducible results

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.
Reply With Quote
  #3  
Old 09-18-2009, 03:12 PM
Zagami's Avatar
Ph.D Doctorate
Points: 4,251, Level: 44 Points: 4,251, Level: 44 Points: 4,251, Level: 44
Activity: 50% Activity: 50% Activity: 50%
 
Join Date: Sep 2009
Location: Tabaka Mission Hospital - Kiisi -Kenya & Italy Sicily
Posts: 165
Thanks: 2
Thanked 28 Times in 24 Posts
Default Re: unreproducible results

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.
Reply With Quote
Reply

Tags
results , unreproducible


Thread Tools
Display Modes

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are On
Pingbacks are On
Refbacks are On

Forum Jump

Similar Threads
Thread Thread Starter Forum Replies Last Post
Human Cytome Project - Update 24 Jan. 2005 Peter Van Osta Cell Biology and Cell Culture 1 08-01-2010 02:18 PM
The Mind Portal...Mapping out the Molecular Biological processes! Jozen-Bo General Science Questions and Layperson Board 3 08-01-2009 11:05 AM
Human Cytome Project - Update 6 Jan. 2005 Peter Van Osta Cell Biology and Cell Culture 0 01-06-2005 11:18 AM
Wave Function Manipulation Experiments Consc Physics Forum 0 12-27-2004 11:07 PM


All times are GMT. The time now is 03:12 AM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.16672 seconds with 16 queries