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The aim was to develop and validate an assay for measuring autoantibodies against human intrinsic factor (IF). For this purpose 1.25pmol of recombinant IF was coupled to each well. Samples (100μL of plasma diluted 1:1) were incubated for 1h, followed by 30min with the detection reagent (biotinylated IF, 3.3pmol/mL). Samples were obtained from healthy blood donors (n=141) and patients with suspected vitamin B  12  deficiency (n=355). The initial assay results (n=99 samples) were in reasonable agreement with those obtained using a commercial assay (Diagnostic Products Corporation). All donors but one showed a negative result. For the patient populations the fraction of positive results decreased with increasing levels of serum vitamin B  12 : 0.67 for &lt;100pmol/L (n=13), 0.17 for 100–150pmol/L (n=23), 0.06 for 151–200pmol/L (n=65) and &lt;0.01 for &gt;200pmol/L (n=254). The imprecision was 11% as judged by repeat analyses. All samples remained positive when the catching reagent was exchanged with vitamin B  12 -saturated IF, suggesting that the samples contained both blocking (type I) and non-blocking (type II)antibodies. Results obtained with IF as the detection reagent correlated to those obtained when anti-immunoglobulin G was used instead. In conclusion, an ELISA using recombinant IF as both catching and detection reagent seems suitable for the detection of IF autoantibodies in plasma.

Autoantibodies against intrinsic factor (IF) measured with an ELISA using recombinant human IF as both catching and detecting reagent