Development of Novel, Ultrasensitive and Practical Enzyme Immunoassay for Anti-Thyroglobulin Autoantibodies

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A number of experiments were carried out to develop a practical and ultrasensitive enzyme immunoassay for anti - thyroglobulin IgG in serum. 1) By testing some variations of immune complex transfer enzyme immunoassay, the following version was found to be the most sensitive and practical. Anti - thyroglobulin IgG in serum was reacted simultaneously with 2, 4 - dinitrophenyl - thyroglobulin and thyroglobulin - beta - D - galactosidase conjugate. The complex formed consisting of the three components was trapped onto polystyrene balls coated with antiN-2, 4 - dinitrophenyl group) IgG. After washing, the complex was eluted from the polystyrene balls with epsilonN-2, 4-dinitrophenyl - L - lysine and transferred to polystyrene balls coated with (anti - human IgG gamma - chain) IgG. B - D - Galactosidase activity bound to the last polystyrene balls was assayed by fluorometry. 2)NSulfosuccinimidyl - 6 - [6 - ((2, 4 - dinitrophenyl) amino) hexanoylamino] hexanoate was synthesized and used to in … More troduce 2, 4 - dinitrophenyl groups into thyroglobulin molecules more easily than before. 3) Anti - thyroglobulin IgG in serum was reacted with thyroglobulin labeled with europium ion, which can be detected more quickly than enzymes. The complex formed was trapped onto polystyrene balls coated with (anti - 2, 4 - dinitrophenyl group) IgG. After washing, the complex was eluted from the polystyrene balls with epsilon N-2, 4 - dinitrophenyl - L - lysine and transferred to polystyrene balls coated with (anti - human IgG gamma - chain) IgG. Europium ion bound to the last polystyrene balls was measured by time - resolved fluorometry. This assay was simpler than the above assay, although less sensitive with larger assay variations. 4) In the above assay with beta-D - galactosidase, the background in the presence of excess of thyroglobulin was unexpectedly high with some serum samples. This interference was suspected to be due to the presence of anti - beta - D - galactosidase antibodies in test serum. and eliminated by using inactive beta-D - D - galactosidase, making it possible to detect anti - thyroglobulin IgG in almost all healthy subjects as well as in all patients with Graves' disease and chronic thyroiditis. Less