As synthetic ACTH and its biologically active or inactive fragments have become available, it was thought that it could be used as a model for the investigation of structural features on immunogenicity and antigen-antibody interactions, and also on the relationship between biological and antigenic properties of the molecule. The synthetic preparations of ACTH-peptides used in this investigation were: (a) (3 1-39 ACTH, which corresponds to the natural pig full chain ACTH and has full biological activity3; (b) p 1-24, a biologically active fragment4; (c) p 1-10; and (d) p 11-24, fragments derived from the preparation of p 1-24 corticotrophin and without biological activity. We would like to thank Dr. R. Schwyzer, of the Department of Molecular Biology, Zurich, for the gift of these preparations.Four groups of six to eight guinea-pigs were injected with different preparations of ACTH, as described elsewhere2. The guinea-pigs received five doses of 1 mg of either natural Ax ACTH (a gift from Prof. Tausk, Organon, Oss, Holland), or synthetic p 1-39 or p 1-24 ACTH. Controls were injected with Freund's adjuvant alone. The strengths of the antisera produced were compared by their ability to bind p l-39-131I in the radioimmuno-logical system described previously2. The results shown in Fig. 1 indicate that p 1-39 was as potent in eliciting antibody formation as was the preparation of natural origin, but p 1-24 was scarcely antigenic. No binding of p l-24-131I by the sera from the guinea-pigs injected with p 1-24 could be detected, thus eliminating the possibility that such sera may contain specific antisera for p 1-24, which would not bind p l-39-131I.Table l. DISPLACEMENT OF ACTH-131I FROM ANTIGEN-ANTIBODY COMPLEX BY 100 NG/ML. OP DIFFERENT PREPARATIONS OF ACTH OR FRAGMENTSOF ACTH PreparationA x ACTH (natural, Organon) p 1-39 (synthetic) P 1-24 P 1-10 p 11-24 Percentage of displacement100 20015-33 (occ. 100) 0 0-5-2Fig. 1. Relative strengths of antisera produced by guinea-pigs against various ACTH preparations. The strengths of the antisera are expressed as the percentage binding of a fixed quantity of p l-39-131I by the same dilution (1/500) of antisera produced in a series of guinea-pigs by the injection of the preparations of ACTH shown in the figure. The control animals were injected with saline in place of hormone Unlabelled ACTH and labelled ACTH compete for binding to antibodies against ACTH1. This is the basis for a radioimmunoassay. The specificity of the antibodies for ACTH can thus be examined by comparing the binding of the labelled species in the presence or absence of different unlabelled species when the antibody is at chain ACTH. Smaller fragments p 1-10 and p 11-24 do not compete in any appreciable degree for the antibody sites.The best known and best studied of the biological effects of ACTH are the adrenotropic and peripheral lipolytic activities. Lebovitz and Engel5 have shown that the same minimum structure is necessary for both activities. In order to verify that the binding of ACTH to its antibody corresponds to a loss of the biological activity of the hormone, it was decided to investigate the Lausanne, effect of ACTH antiserum on the in vitro lipolytic activity Switzerland, of p 1-39 and p 1-24 synthetic corticotrophin. The method used was essentially that of Lebovitz and Engel6, with the exception that glycerol was measured rather than FFA (free fatty acid), as glycerol is not recycled. After 1 h preincubation and 3 h incubation in 3 ml. Krebs-Ringer bicarbonate buffer containing 2 per cent albumin and the substances the effects of which on lipolysis are to be tested, the extent of lipolysis was determined by measuring the medium glycerol concentration in each flask by the method of Wieland7. The results (Table 2), which are expressed as micromoles glycerol/g tissue/3 h incubation, indicate the dependency of the glycerol production of epididymal fat pads on the ACTH concentrations. Table 2. LIPOLYTIC EFFECT OF SYNTHETIC p 1-39 //moles glycerol/g Additions to medium tissue/3 h incubationNone25 ng/ml. p 1-39 100 ng/ml. p 1-39 250 ng/ml. p 1-39 5-041-61 (6) 8-162-28 (6) 10-53208 (6) 12-833-45 (6) Tissues were incubated for 3 h at 37 C in Krebs-Ringer bicarbonate medium containing 2 per cert beef serum albumin in an atmosphere of 95 per cent oxygen : 5 per cent carbon dioxide. Results are expressed as the medium glycerol concentration after incubation per g tissue per 3 h of incubation and are given as meanstandard deviation. The figures in parentheses are number of observations. Table 3. EFFECT OF INCUBATION WITH ANTISERUM ON LIPOLYTIC ACTIVITY preserOF 6 1-39 AND p 1-24 //moles glycerol/g Additions to medium tissue/3 h incubationHISTOLOGY (a)p 1-39 None100 ng/ml. p 1-39 100 ng/ml. p 1-39 + antiserum 1/3,600 Antiserum 1/3,600(b) P 1-24 None 100 ng/ml. p 1-24100 ng/ml. p 1-24 + antiserum 1/3,600 Antiserum 1/3,600 4-400-71 (6) 14-200-71 (6) 8120-90 (8) 4-591-25 (6) 3-04 + 0-57 (5) 8-200-94 (8) 6-090-69 (5) 3-310-14 (4) All media were allowed to stand at 4 C for 1 h before adding tissue. The tissue had been pre-incubated for 1 h. Incubation in the media was for 3 h. Rlt d th di ll cocentration after incubation per g tissue per 3 h of incubation and are given as meanstandard deviation. The figures in parentheses are number of observations. The effects of antibodies on the biological activity of both the 1-39 and 1-24 poly-peptides are shown in Table 3. While non-specific effects of antisera were found at low dilution, this was not the case at the dilutions used in these experiments. It is interesting to see that the antibodies obtained from guinea-pigs injected with the 1-39 synthetic peptide block the activity not only of the complete poly-peptide but also of the 1-24 fragment. This finding establishes a relationship between activity inhibition and radio-immunological binding.To sum up, these experiments show that the synthetic full chain p 1-39 ACTH is antigenic, whereas the 1-24 fragment such a limiting concentration that competition occurs. The has only little, if any, immunogenic activity. It behaves results of such experiments are given in Table 1. It can as a hapten and binds to antibodies produced against be seen that p 1-24 binds to the antibodies to p 1-39, the complete ACTH chain. Binding to antibodies is although it produces less displacement than the full followed by the loss of in vitro lipolytic activity of the y full chain or the 1-24 fragment. This work was supported by a grant from the Fonds National Suisse de la Recherche Scientifique.