Microtiter plate assay for biopterin using cryopreserved Crithidia fasciculata

This chapter presents microtiter plate assay for biopterin, using cryopreserved Crithidia fasciculate. The term “total biopterin” includes biopterin, dihydrobiopterin, and tetrahydrobiopterin. Most of the diagnostic criteria and normative data available for the Crithidia assay have been obtained by automated tube assay without chloramphenicol. This requires heating to remove protein, and although heat treatment is not necessary for the microtiter plate assay, the practice has continued for comparative, diagnostic purposes. Plasma samples are diluted 1:30 and whole-blood samples are diluted 1:40 in phosphate buffer. Dried blood spots have an 8-ram circle punched out into 1 ml of phosphate buffer. Samples are then heated in a steamer for 10 min, centrifuged, and passed through 0.2-μm filters to clarify. When there are reasons to suspect that the biopterin level is raised, for example, in phenylketonuria, further dilutions should be prepared in phosphate buffer after filtration.