New enzyme immunoassay for detecting total, type I, and type II intrinsic factor antibodies.

Abstract

SUMMARY A method for the detection of total, type I, and type II intrinsic factor antibodies was devised. The technique comprises a two-site solid phase enzyme linked immunosorbent assay (ELISA), with human intrinsic factor conjugated with horseradish peroxidase as label and attached to polystyrene tubes as solid phase. One conjugation provides sufficient material to assay more than 10 000 patient samples. The label proved stable during the course ofthis evaluation and was still in use more than 12 months after preparation. When applied to 45 serum samples from cases of pernicious anaemia, intrinsic factor antibodies were shown in 30 (67%). Simplicity, high capacity, low cost and label stability, combined with relatively high clinical sensitivity make the method suitable for cost effective screening of large numbers of samples. Simple modifications to the basic assay reagents permitted type I and type II intrinsic factor antibodies to be differentiated.Despite being available for almost 30 years,' application of tests for circulating antibodies to gastric intrinsic factor has been limited, because the tests have not been readily available to all routine laboratories, and secondly, they have been capable of showing the presence of antibodies in only about 50% of patients with pernicious anaemia. It has been proposed, however, that the demonstration of intrinsic factor antibodies in patients with megaloblastic anaemia and low serum vitamin B12 concentrations could be regarded as diagnostic ofpernicious anaemia and may avoid the need for B12 absorption tests.2Two types of intrinsic factor antibody are known to exist,3 one ofwhich reacts with the intrinsic factor-B12 binding site (type I); the other (type II) detects an antigen site unrelated to the B12 binding activity. Methods of detection have concentrated on type I, making use ofthe ability ofthe antibody to prevent the uptake of 57Co-B12 by intrinsic factor, combined with a charcoal separation procedure.' Recently, a method that used 1251-labelled intrinsic factor has been described: this focuses on the ability of the antibody to bind to intrinsic factor directly' instead of showing a type I antibody blocking effect.' This method has also been modified to detect and quantify both type I and type II antibody.89 A commercial intrinsic factor antibody method [Walker

Laboratory assessment of vitamin B12status

“…Two types of plasma-intrinsic factor antibodies have been detected in the plasma of >60% of patients with pernicious anaemia44 51 with type I blocking the binding of B 12 to intrinsic factor and type II stopping the attachment of intrinsic factor or intrinsic factor-B 12 complex to ileal receptors. Achlorhydria may be suspected by the presence of raised gastrin levels 52…”

Limited value of testing for intrinsic factor antibodies with negative gastric parietal cell antibodies in pernicious anaemia: Figure 1

“…Antibodies to intrinsic factor are seen in 50–70% of patients and are viewed as a more specific marker of pernicious anaemia 3. The necessity of early detection of gastric parietal cell and/or intrinsic factor antibodies (IFA), with the need for regular follow-up of pernicious anaemia patients to watch for the development of additional autoimmune diseases has been recently highlighted 4.…”

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

“…These assays are only able to identify type I autoantibodies, most often referred to as blocking antibodies because they are able to block the uptake of vitamin B 12 by IF. More recently, ELISA employing IF as both the catching and the detection reagent was also reported (8). In addition, false-positive results have been reported in patients with high serum vitamin B 12 levels (6).…”

“…In the present paper we further developed the concept to use IF as both catching and detection reagent (8) and we present an assay based on commercially available recombinant human IF.…”