A synthetic 59-base oligonucleotide containing a C12 modifier was activated with excess disuccidimidyl suberate
Examples of such renewable dicarboxylic acids are dimethyl suberate
, dimethyl sebacate, or dimerized (un)saturated fatty acids.
, sebacate, dodecanedioate, and terephthalate) dissolved in pyridine to form complexes (36) similar to those reported by Maritz (37) for copper, zinc, cadmium, and nickel adipates.
AZXMP was coupled to HRP by use of the homobifunctional reagent disuccinimidyl suberate
(DSS), which incorporates an 8-carbon chain linker.
Among these inborn errors are glutaric aciduria type I (18,19) (glutarate concentrations may be within reference values, whereas 3-hydroxyglutarate is present); medium-chain acyl-CoA dehydrogenase deficiency (adipate, suberate
, and sebacate concentrations may be within reference values, but the presence of suberylglycine and hexanoylglycine will reveal the disorder) (20); multiple acyl-CoA dehydrogenase deficiency, particularly in its mild forms (metabolites suggesting such a disease, including ethylmalonate and glutarate, are quite variable); and 2-ketoglutarate dehydrogenase deficiency (2-ketoglutarate excretion ranges from within reference values to 10 times higher than the upper limit of the reference interval).
For the cross-linking experiments, bis-sulfosuccinimidyl suberate
(BS3) was added as a cross-linker to the serum sample before immunoprecipitation.
(Pierce) was dissolved to 28 g/L in dimethyl formamide (Aldrich), and 22 [micro]L was added to the capture probe and incubated at room temperature for 10 min.
Covalent attachment of the mecA capture probe (5'-GTCATTTCTACTTCACCATTACCAAC-3') was accomplished by reacting the homobifunctional cross-linker disuccidimidyl suberate
(Pierce) with the 3' amine of the capture probe and amines on the biosensor surface, producing a stable amide linkage.