FREE AND BOUND BIOTIN MOLECULES IN HELMINTHS - A SOURCE OF ARTIFACTS FOR AVIDIN BIOTIN-BASED IMMUNOASSAYS

The avidin-biotin molecular recognition system is widely used in paras ite immunology. However, the presence of biotin and/or biotin-containi ng molecules (BCMs) in samples may lead to erroneous results, In the w ork reported herein we investigated the extent to which biotin and BCM s present in helminth extracts may interfere in avidin/biotin-based im munoassays and developed an enzyme-linked immunosorbent assay (ELISA) for quantification of these components. In avidin-based ELISA using an tinematode monoclonal antibodies, an extract of the nematode Anisakis simplex- showed very high background reactivity due to biotin/BCMs, wh ereas the background reactivity in an extract of the nematode Trichine lla spiralis was negligible. To investigate interspecies differences f urther, we performed Western-blot analyses (with avidin as the detecto r) of extracts from seven nematodes (A. simplex, Ascaris suum, Toxocar a canis, Hysterothylacium aduncum, T. spiralis, and Trichuris muris) a nd the cestode Bothriocephalus scorpii. Even within superfamilies ther e was considerable variation in the banding patterns obtained. The abo ve-mentioned results confirm that biotin and BCMs may be a significant source of interference in ELISA and immunoblotting, two of the techni ques most widely used in parasitological immunodiagnosis. A competitio n ELISA designed to allow accurate quantification of biotin and BCMs i n helminth extracts likewise indicated very considerable interspecies variation. Both A. simplex and H. aduncum had very high biotin/BCM con tents. Microdialysis of extracts in the presence of dimethylsulfoxide to remove free biotin prior to ELISA indicated that the high biotin/BC M content of the H. aduncum extract (but not the A. simplex extract) w as very largely due to free biotin. Taken together, these results indi cate that extreme caution should be exercised in the use of avidin/bio tin-based immunoassays for the detection of helminth antigens and that in many cases it may be better to use an alternative recognition syst em.