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Technical Literature

The Intrinsic Factor IgG test is a rapid ELISA method for the detection of antibodies to intrinsic factor, a protein involved in the transport of vitamin B12. The components of the test are for in vitro diagnostic use only.

Intrinsic Factor (IF) is an essential glycoprotein involved in the transport of vitamin B12 across the intestinal mucosa. This protein binds to the vitamin forming a complex, which permits B12 absorption into the bloodstream. Vitamin B12 is necessary for the maturation of erythrocytes and a deficiency leads to the development of anaemia.

Failure to produce or utilise IF results in pernicious anaemia. Autoantibodies to IF act by either blocking the formation of the IF-B12 complexes or by binding to other sites on the complexes, preventing absorption.

The determination of serum levels of anti-IF antibodies provides a means to differentiate between autoimmune pernicious anaemia, non-autoimmune pernicious anaemia (e.g. atrophic gastritis and pancreatic exocrine insufficiency) and other forms of vitamin B12 related anaemia.

Principle of the Intrinsic Factor ELISA Test
Diluted serum samples are incubated with recombinant Intrinsic Factor immobilised on microtitre wells. After washing away unbound serum components, rabbit anti-human IgG conjugated to horseradish peroxidase is added to the wells, and this binds to surface-bound antibodies in the second incubation. Unbound conjugate is removed by washing, and a solution containing 3,3’,5,5’-tetramethylbenzidine (TMB) and enzyme substrate is added to trace specific antibody binding. Addition of stop solution terminates the reaction and provides the appropriate pH for colour development. The optical densities of the standards, controls and samples are measured using a microplate reader at 450nm. Optical density is directly proportional to antibody activity in the sample.


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