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The page below is a sample from the LabCE course Vitamin D. Access the complete course and earn ASCLS P.A.C.E.-approved continuing education credits by subscribing online.

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Immunoassays

Most immunoassay methods can separate and quantify vitamin D2 and vitamin D3. But, depending on the specificity of the antibody used in the immunoassay method, some immunoassays measure only one form.
Several Food and Drug Administration (FDA)–approved immunoassay tests are available, including quantitative chemiluminescent immunoassay (CIA) methods.
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.

CLIA Vitamin D assay example