Triiodothyronine methyl ester hydrochloride (T,•MEH) was obtained by acid methanol method of Ashley and Harington. T,.MEH was conjugated to bovine serum albumin (BSA) by carbodiimide method of Oliver et al. 3.2 mg of T,.MEH-BSA complex was dissolved into 0.8 ml of sterile physiological saline solution. This solution was emulsified in equivalent volume of complete Freund's adjuvant and injected into toe-pads of rabbits (0.4 ml per injection). Rabbits were boosted with intramuscular injections of

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... 1 mg of antigen in complete adjuvant every month. Sera were obtained one week after the last injection one year later. a) T, in 0.1 ml of unknown serum or standard T3 added to the serum of cretinism was extracted with 2 ml of methanol. Extraction rate was 85+2%. After evaporation of methanol, extracted T3 was dissolved into 1.0 ml of barbital buffer (pH 8.6, 0.05 M) containing BSA in 0.5%. b) Because of binding of the "5I-T, to serum protein, direct assay of serum yielded high nondisease related T3 values. In order to inhibit such binding, 0.01 ml of dinitrophenol (DNP, 1.20 x 10' M) or diphenylhydantoin (DPH, 5.85 x 10' M) was added to 0.1 ml of crude serum diluted with 0.89 ml of BSA diluent. To each assay system in a) or b), 0.1 ml of 125I-T, in BSA diluent (less than 25 pg, specific activity 70-450 pCi/pg, Abbott or Mallinckrodt) and 0.1 ml of antiserum diluted with BSA diluent (1: 1,000-2,000) were added. This gave a total volume of 1.2 ml in each assay system. The mixtures were incubated at 5°C for 18 hours. Then 0.2 ml of dextran-coated charcoal (Charcoal 5.0 g and dextran 0.5 g in 400 ml of water) was added to them. One hour after shaking at room temperature for 5 min., mixtures were contrifuged at 3,000 r.p.m. at 5°C for 15 min. and then radioactivities of supernatant and precipitate were counted in a well-typed scintillation counter. Percentage of bound 19 -113 was calculated. The ability of various T, analogues to inhibit the binding of 125I-T, to antibody was compared to that of T, (1.00) with the following results : 1-thyroxine, 0.0021; triiodothyroformic acid,0.066; triiodothyropropionic acid,0.081; triiodothyroacetic acid,0.061; tetraiodothyroformic acid, 0.00014; tetraiodothyroacetic acid, 0.0013; 1-monoiodotyrosine, 0.000070 and 1-diiodotyrosine, 0.000059, respectively. Mean T, values in methanol extraction and DPH addition method in euthyroid, Vol. 48 No. 8