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

Abstract

The aim was to develop and validate an assay for measuring autoantibodies against human intrinsic factor (IF). For this purpose 1.25 pmol of recombinant IF was coupled to each well. Samples (100 microL of plasma diluted 1:1) were incubated for 1 h, followed by 30 min with the detection reagent (biotinylated IF, 3.3 pmol/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 <100 pmol/L (n = 13), 0.17 for 100-150 pmol/L (n = 23), 0.06 for 151-200 pmol/L (n = 65) and <0.01 for >200 pmol/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.