Quantitative inhibition ELISA for diagnosis of schistosomiasis using human IgG labeled with fluorescein and anti-fluorescein/peroxidase

Abstract

Usually mouse monoclonal antibodies are used in inhibition assays for antibody determination. Interference may occur in these inhibition assays due to presence of naturally occurring anti-mouse antibodies in some human serum samples. To avoid such interference, human IgG isolated from a pool of serum samples of S. mansoni patients and highly positive for IgG against S. mansoni soluble egg antigen (SEA) was used in inhibition ELISA for diagnosis of S. mansoni infection. The assay was based on inhibition of binding of human IgG labeled with fluorescein to S. mansoni SEA coating microtitration plates by tested serum samples. Plates were washed and labeled human IgG reacted with SEA was linked to peroxidase enzyme by incubation with anti-fluorescein/peroxidase conjugate. The assay showed 90% sensitivity and 96.3% specificity. The level of inhibition in ELISA showed highly significant positive correlation with stool egg output (Kandall's tau b = 0.512, P < 0.001). To make the assay quantitative, serial dilutions of the highly positive human serum pool, used for preparation of human IgG, were applied in each plate and concentration of anti-SEA antibodies in serum samples tested was calculated from a 4-parameters logistic curve equation. The highly positive serum pool used as a standard was considered to contain one million arbitrary units of immunoglobulins against S. mansoni SEA. Human IgG is expected to be more practical in inhibition assays than mouse monoclonal antibodies due to elimination of interference caused by naturally occurring human anti-mouse antibodies. Also, large amount of human IgG could be purfied from remnants of serum samples highly positive for the proposed antibodies. A higher specificity and sensitivity could be obtained if IgG is isolated by affinity purification instead of ammonium sulphate precipitation. In conclusion, human IgG isolated from highly positive serum samples could be used in sensitive and specific diagnostic antibody determination inhibition assays for diagnosis of infectious and autoimmune diseases.