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

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.

Mike Zannelli, Assay Development
Zannelli at Endogen.com


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