ELISA, high background from serum with no Ag - Help needed please!! (Mar/12/2008 )

Hi guys,

I'm doing ELISA to detect Abs induced in mice upon immunization. My problem is I get high background from mouse serum even when I don't use my protein in coating, I only use carbonate buffer to coat in this case.
So, I coat my plate with in house purified protein at 4ug/ml concentration. I also include some wells without protein (carbonate buffer only). When I use serum at the same dilution let's say diluted 1:100 on both wells (protein coated and no protein), I get high signal from no protein wells. However, my positive control which is purified monoclonal antibodies gives no signal without protein.
I think it's the serum, heterophilic antibodies may be? I don't know.

I use 5%BSA-TBS with 0.05% tweeen-20 as my blocking buffer and diluent for serum samples. I wash with the same thing but no BSA.
My friend told me to change my blocking buffer to 5% milk? Is it going to help?
Some people are talking about preincubation of serum with blocking buffer? Is that ok? Or it will affect my Ab of interest/

Could anyone tell me is there is any suggestion?

Thanks
Anwar

hi,

Do you have the same background levels using serum hiperimmune or serum preinmune as a primary antibody?
If the answer is yes, you can have a problem with the blocking buffer (percentage, unespecific protein used) and antibodies from serum could bind to the polystirene wells.
If the answer is no, you may have a cross reactive response in antibodies of hyperimmune serum against BSA used in blocking buffer. You must change the protein unspecific that you use on the block.

On the other hand, a 1/100 dilution of the serum can give you enough background noise, has tested whether the background noise persists when primary serum is diluted more times?.

I hope you serve

Hey Tonix,

Yes, the background is almost the same in my preimmune sera and sera after immunization (no significant difference).
I didn't try higher dilutions with primary Abs (sera) to see if the background persists or not. I will give it ashot tomorrow. Also, I'm gonna try different blocking buffers and different percentage.
The problem with higher dilutions is that I don't expect to see any response with high dilutions (Ab response is week).

Thanks

Hi anwar,
I observed that if we take either hyperimmune or preimmune sera of mice, 1/100 dilution will give very high background in ELISA
so you should never use 1:100 dilution serum. You can start only from 1:500
all the best

Hi Anwar,

This is a very common problem for ELISAs because of 2 issues. First there is a high IgG content present in serum which provides significant background especially when low dilutions are used. Preimmune sera typically have lower IgG contents because young animals have not built up significant humoural responses until they get older. Even sera from an unimmunized animal will show increased ELISA signals that are non-specific over a typical immunization period of 10-12 weeks. Polystyrene plates typically used for ELISA bind significant amounts of protein and so the use of sera at low dilutions inevitably results in adsorption of detectable amounts of non-immune IgGs. Secondly, depending on the immune titre, you may or may not be able to measure specific reactions over and above the background. My company, Larial Proteomics develops high fidelity custom immunologicals for clients. We typically use biosensors to measure serum antibody interactions with antigens because we obtain much greater specificity with kinetically driven reactions whose time courses are in the millisecond range. The other important point I would make is that ELISA specificity can be significantly improved by affinity purification of the immune globulins. We generally don't bother to conduct ELISAs with serum because of the problem you have described. Instead we build an affinity purification platform based on data we obtain from biosensor measurements then test antibodies isolated by affinity chromatography by ELISA. It generally takes 2-3 affinity purifications to remove all of the non-specific IgGs from preparations. If you would like to contact me regarding this or other issues please fell free to call 416-673-8155 or toll free 877-873-8155.

Sincerely,

Jeffrey H.M. Charuk, Ph.D.,
Chief Scientist,
Larial Proteomics Inc.