Homogeneous enzyme immunoassays using avidin-ligand conjugates

2 Homogeneous enzyme immunoassays using avidin-ligand conjugates Interaction of avidin (or haptenated avidin) with 

Homogeneous competitive enzyme immunoassays using substrate-labeled antigen The method of Ngo et al. (1981) to 

Homogeneous enzyme-immunoassay with avidin-ligand as enzyme modulator 

Pyruvate carboxylase (EC 6.4.1.1 from Bacillus stearothermophilus), which is not present in significant amounts in human body fluids, is grown and purified as described by Bacquet and Twumasi (1984). In the following assay, S,S-diphenylhydantoin (DPH) is used as the ligand. 

The reaction mixture (I ml of 100 mM Tris-HCI buffer, pH 8.0) contains 5.0 /imol MgCb, 0.1 mmol KHCO3, 3.3 imol sodium pyruvate, 0.1 /imol acetyl coenzyme A, 1.7 pmo adenosine 5 -triphosphate, 0.1 /imol dithiobis(2-nilrobenzoic acid) (DTNB) and 1.0 U of citrate synthase. The activity of pyruvate carboxylase is determined after adding the enzyme from the initial increase in absorbance due to the reduction of DTND by coenzyrae A (molar extinction coefficient DTNB at 412 nm is 14150 M em ). 

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