ELISA Assay

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NathF
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ELISA Assay

Hello Everybody,

Please does anyone know how I can eliminate non-specific binding in an ELISA (Sandwich Method) to assay for Ab in cell culture?

Thank you

NathalieHV
NathalieHV's picture
As discussed in another topic

As discussed in another topic, you can use Tween 20. However, if you do cell based ELISA (for surface molecule detection) it may increase your background because it permeabilizes your cells.
Nathalie HV

tittletv
tittletv's picture
You might want to try

You might want to try blocking with 1% gelatin. Gelatin is non-immunogenic in most animals and it is as good as, if not better than, using Tween.

MadScientist
MadScientist's picture
NathF wrote:Hello Everybody,

NathF wrote:

Hello Everybody,

Please does anyone know how I can eliminate non-specific binding in an ELISA (Sandwich Method) to assay for Ab in cell culture?

Thank you

There is a plethora of blocking agents that you can use for your ELISA. Typically the most commonly used ones are casein (or milk powder for a cheaper alternative!), BSA, Tween20, gelatin, etc.

Typically 1% solution of the blocking agents should be more than enough to reduce non-specific binding. If you're still having problems, then try increasing the blocking solutions concentration to 2%, although it is most likely that this will not be neccessary.

Hope this helps!

Scot E. DOwd Ph.D.
Scot E. DOwd Ph.D.'s picture
try using different species

try using different species for your capture and detection antibodies. e.g. sheep may produce more non-specific than rat. They quality of the antibodies for detection are very important. ALso monoclonal vs polyclonal is important aspect.

Dr. R.P. Singh
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NathF wrote:Hello Everybody,

NathF wrote:

Hello Everybody,

Please does anyone know how I can eliminate non-specific binding in an ELISA (Sandwich Method) to assay for Ab in cell culture?

Thank you

You may also try lactalbumen hydrolysate (LAH) 1-3% if very efficient blocking is required as in the case of use of poly clonal sera.
R.P.Singh

samm
samm's picture
You can try 0.7% gelatin and

You can try 0.7% gelatin and 0.05% Tween 20. Also, ensure that your secondary Ab is good - Jackson Immunoresearch has some really good preadsorbed ones.

Fujiwara
Fujiwara's picture
Check also the plate used.

Check also the plate used. Some brand of plates are likely to have some background. We use a Maxisorp (for antibody coat) or Polysorp (for antigen coat), both from Nunc. Using these plates we decreased the background to the blank levels.
Since you are trying a sandwich ELISA, the Maxisorp will work better in this case. It worthy a try.

Teia
Teia's picture
Hey,

Hey,
Can anyone suggest which tween - 20 I should use? There are about 10 different kind in Sigma. Any catalog numbers?
Thank you in advance

ronpbl
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Teia wrote:Hey,

Teia wrote:

Hey,
Can anyone suggest which tween - 20 I should use? There are about 10 different kind in Sigma. Any catalog numbers?
Thank you in advance

We use enzyme grade Tween 20 in the production of our ELISA kits from Fisher (cat #BP337-500). I am sure other vendors sell a comparable product. Just look for a high quality prep like enzyme grade or protease-free grade.

JenniferGeiger
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Hello there. I just joined

Hello there. I just joined this forum and I hope I am posting this correctly. I am an assistant microbiologist in a lab that performs IgM antibody capture ELISAs on sentinel chicken blood. We are testing for west nile virus and eastern equine encephalitis virus. This season we have had consistent trouble with one particular chicken's serum. Let me run thru our process.
1) heat inactivate chicken serum (so as not to worry about possibly infectious virus becoming airborn)
2) coat plates with goat anti-chicken IgM and coating buffer in a 1/3000 dilution which incubates overnight at 4 C
3)remove coating dilution and apply blocking buffer..incubates at rm temp for 1/2 hour
4) wash plates then add 1/400 dilutions of serum in wash buffer and positive and negative controls in wash buffer...incubates at 37C for 1 hour
5) wash plates then apply antigen and mock antigen diluted 1/1000 in wash buffer...incubates overnight at 4C
6) wash plates then apply conjugate diluted 1/2500 in blocking buffer...incubates 1 hour at 37C
7) wash plates then apply ABTS substrate...plates incubate at rm temp, in the dark until coloration forms
Now....question....for about 7 weeks now we have consistently seen the wells for our #2 chicken show coloration. The P/N value is not a positive. however..the coloration is very obvious...compared to the background. However....there is also coloration in the Mock Ag lanes. Could this be due to factors in this particular chicken's blood? Are we getting this affect because we heat-inactivate the serum or is there something we are over-looking?

Add colour 2 ur life
Add colour 2 ur life's picture
hi there

hi there
first thing is to reduce non specificity u need to choose the secndry antibody of minimal cross reactivity for that jackson is providing the minimal cross reactive ab
and also try with some other blocking reagents like casein albumin choose also skim milk powder if u r secn is not belongs to goat because milk also have some immunoglobulins
and try with adding detergents like tween 20 (pbst)buffer
All the best

JenniferGeiger
JenniferGeiger's picture
Add colour 2 ur life wrote:hi

Add colour 2 ur life wrote:

hi there
first thing is to reduce non specificity u need to choose the secndry antibody of minimal cross reactivity for that jackson is providing the minimal cross reactive ab
and also try with some other blocking reagents like casein albumin choose also skim milk powder if u r secn is not belongs to goat because milk also have some immunoglobulins
and try with adding detergents like tween 20 (pbst)buffer
All the best

Hello and thank you for the advice. Perhaps we could try the secondary antibody from Jackson. However, we already are using casein...it is in our blocking buffer, as well as Tween 20. Tween 20 is also in our wash buffer. I just think that it is odd that these strange results are only occurring with one chicken's serum. The rest of the plate "behaves" as you would expect...

geet
geet's picture
u can use himedia tween 20

u can use himedia tween 20 whose refrence no is RM156-500gm.hope this will work