A sandwich method of enzymoimmunoassay. I. Application to rat and human alpha-fetoprotein.
An enzymoimmunoassay (EIA) for the quantitation of soluble antigens having at least two antibody-combining sites is descirbed. This non-competitive sandwich method comprises three steps: 1) the antigen to be assayed is reacted with the antibody-coated cellulose immunosorbent, 2) the enzyme-labelled antibody is then incubated with the antigen bound to the solid phase, 3) the enzymatic activity of the immunosorbent is then measured. This activity increases with the quantity of antigen to be assayed. Examples of the application of this method to the assay of rat and human alpha-fetoprotein (AFP) are given. When applied to rat and human AFP, this assay gives reproducible results in the range of 10--1000 ng/ml and 3-1000 ng/ml respectively. AFP sera concentrations of normal and pregnant rats were assayed by EIA, radioimmunoassay RIA and rocket-immunoelectrophoresis (RIE). In all the cases good agreement was noted among these three techniques. Rheumatoid factor, whenever present, may interfere with the assay. However, the effect of this interaction can be eliminated. The advantages of the EIA method can be listed as follows: a) no pure antigen is required, b) the final color reaction is developed from the solid phase. This feature eliminates most non-specifically interfering factors, c) the range of the assay covers a 2 log-scale, d) only inexpensive equipment is used, e) results are obtained within 24 hr, f) sensitivity and reproducibility lie within a range comparable to that of RIA.
D000818:Animals; D000906:Antibodies / Q000302:isolation & purification; D001666:Binding Sites, Antibody; D002855:Chromatography, Thin Layer; D005260:Female; D005326:Fetal Proteins / Q000302:isolation & purification*; D005949:Glucose Oxidase / Q000378:metabolism; D006801:Humans; D007106:Immune Sera / Q000302:isolation & purification; D007114:Immunization; D007118:Immunoassay / Q000379:methods; D007122:Immunoelectrophoresis; D011247:Pregnancy; D011817:Rabbits; D011863:Radioimmunoassay; D051381:Rats; D013997:Time Factors; D000509:alpha-Fetoproteins / Q000032:analysis / Q000276:immunology / Q000302:isolation & purification*
D000906:Antibodies; D005326:Fetal Proteins; D007106:Immune Sera; D000509:alpha-Fetoproteins; D005949:Glucose Oxidase