ELISA

iG iguana at argo.net
Sat Nov 16 12:05:31 EST 1996


sherry weppler wrote:
> It has now come time to write up the lab report, and we are to include 
> in our discussion some information about capture ELISA and sandwich
> ELISA. 

Hi Sherry,

Of course you know ELISAs can be fashioned in many different ways, but
if you are simply concerned with conjugates other than alk phos, then
here are some Enzyme/Substrate Systems.  If I've missed the mark of your
question, please feel free to email me here or at work.

Enzyme/Substrate Systems:

Horseradish peroxidase (HRP) + Ortho-phenylene diamine (OPD)
Stopping solution: 1.25 M H2SO4
Nonstopped: Green/orange
Stopped: Orange.brown
Read (stopped): 492 nm

Horseradish peroxidase (HRP) + Tetra-methylbenzidine (TMB) 
Stopping solution: SDS (1%) OR 0.18 M H2SO4
Nonstopped: Blue
Stopped: Yellow
Read (stopped): 450 nm

Horseradish peroxidase (HRP) + 2,2'-Azino
di-ethylbenzothiazoline-sulfonic acid (ABTS)
NO Stop
Nonstopped: Green
Read: 414 nm 

Horseradish peroxidase (HRP) + 5-Aminosalicylic acid (5AS)
No Stop
Nonstopped: Brown
Read: 450 

Horseradish peroxidase (HRP) + Di-aminobenzidine (DAB)
No Stop
Nonstopped: Brown
Read: N/A

Alkaline Phosphatase + Para nitrophenyl phosphatate (pnpp)
Stopping solution: 2M sodium carbonate
Nonstopped: Yellow/green
Stopped: Yellow/green
Read (stopped): 405 nm

b-galacosidase + O-Nitrophenyl b-D-galactopyranoside (ONPG)
Stopping solution: 2M sodium carbonate
Nonstopped: Yellow 
Stopped: Yellow
Read (stopped): 420 nm

Urease + Bromocresol
Stopping solution: 1% merthiolate
Nonstopped: Purple
Stopped: Purple
Read (stopped): 588 nm 

We utilize both HRP labelled antibodies as well as biotinylated
antibodies (biotin labelled).  There are many benefits to biotinylated
antibodies.  Streptavidin-HRP is then added -- the biotin-streptavidin
have an strong bond and the substrate, TMB, reacts with the HRP on the
streptavidin.  Then we stop with H2SO4.  Voila, assay for sale.  Of
course I wish it was this easy.

Sincerely,
Mike Zannelli, Assay Development
Zannelli at Endogen.com

-- 
MZ



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