Most immunoassay methods can separate and quantify vitamin D2 and vitamin D3. However, depending on the antibody specificity used in the immunoassay, some immunoassays measure only one form.
Several Food and Drug Administration (FDA)–approved immunoassay tests, including quantitative chemiluminescent immunoassay (CIA) methods, are available.
The procedure for most FDA methods is as follows:
- Step One: 25-hydroxy vitamin D is dissociated from its binding protein and binds to the specific solid phase antibody.
- Step Two: Vitamin D-isoluminol tracer is added, and unbound material is removed with a wash cycle.
- Step Three: The reagents are added to initiate the chemiluminescent reaction.
- Step Four: The light signal is detected by a photomultiplier as relative light units; this measurement is inversely proportional to the concentration of 25-hydroxy vitamin D.
Aside: Washing steps are necessary to remove unbound material and decrease the background, thereby increasing the signal. Insufficient washing will create a high background. Since this assay measurement is inversely proportional to the concentration of 25-hydroxy vitamin D, an increased amount of light will result in an inaccurate decrease in results.
This
video is helpful for an overview of the assay.
