AMPPD phosphate

Substrates for AP are, e.g.,AMPPD (disodium 3-(4-methoxyspi-ro 1 >2-dioxethane-3,2 -ticyclo[3.3.1.1 ] decan -4-yl)phenyl phosphate) or CSPD (disodium 3-(4-methoxyspiro l,2-dioxethane-3,2 -(5"-chloro)tricyclo [3.3.1.1] decan -4-yl)-phenyl phosphate). 

Dioxetane phosphate visualization solution Prepare 0.1 mg/ml AMPPD or CSPD (Applied Biosystems) or 0.1 mg/ml Lumigen-PPD (Lumigen) substrate in dioxetane phosphate substrate buffer (see recipe). Prepare just before use. Lumi-Phos 530 (Boehringer Mannheim or Lumigen) is a ready-to-use solution and can be applied directly to the membrane. 

Various 1,2-dioxetane derivatives have been synthesized and reported. Among them, AMPPD (Adamantyl Methoxy Phenyl Phosphoryl Dioxetane) is a well known dioxetane bearing a spiroadamantyl group at the 3-position and a phenol phosphate at the 4-position, which is now used for chemiluminescence enzyme immunoassay (CLEIA) using alkaline phosphatase with highly sensitive detection. 

For each type of assay, the label or tag involved is listed along with typical substrates, type of absorbance, and type of instrument required. RIA radioimmunoassay IRMA immunoradiometric assay CPM counts per minute ELISA enzyme linked immunosorbent assay EIA enzyme immunoassay TMB 5,5 tetramethylbenzidine HPA p hydroxyphenylacetic acid AMPPD 4 methoxy 4 (3 phosphatephenyl) spiro(l,2 dioxetane) 3,2 adamantane p NPP p nitrophenyl phosphate 4 MUP 4 methylumbelliferyl phosphate ONPG o nitrophenyl P galactopyranoside MUG 4 methylumbelliferyl P D galactopyrano side AMPGD 3 (4 methoxyspiro( 1,2 dioxetane 3,2 tricyclo(3.3.1.1(3,7))decan) 4 yl)phenylgalacto pyranoside ECL electrochemiluminescence. 

Using alkaline phosphatase-labeled antibodies to protein antigens and the meta-phenyl phosphate dioxetane shown in Fig. 41 (referred to as AMPPD or Lumigen PPD), one can conduct sandwich-type immunoassays with a solid phase immobilized capture antibody and an enzyme-labeled signal antibody (B19). After the... 

Replacement of this colorimetric reaction with a chemiluminescent process provides for better sensitivity as well as easier reusability of membranes. The development of compounds, such as adamantyl 1,2 dioxetane phosphate (AMPPD) (1) provides an easy alternative to previous detection schemes as this compound is also destabilized by alkaline phosphatase, resulting in the production of light, which will expose a standard X-ray film. Probes produced by exactly the same methods as for NBT detection are perfectly usable in this process. The membranes with the bound alkaline phosphatase are soaked in a... 

Chemiluminescent substrate Admantyl 1,2-dioxetane phosphate (AMPPD) from Tropix, Bedford, MA. The stock solution is at a concentration of 10 mg/mL. 

FIGURE 5.2 Dioxetane chemiluminescence reaction pathway. A, AMPPD adamantyl 1,2-dioxetane phosphate) when X = H or CSPD when X = Cl B, dioxetane anion C, adamantanone D, methyl-oxybenzoate anion and E, light emission at 477 nm. 

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