56 THE PURIFICATION OF ANTITOXIN BY ABSORPTION OF NON-ANTITOXIC ANTIBODIES. C. G. POPE AND MURIEL F. STEVENS. With technical assistance of J. C. MANUEL. From the Wellcome Research Laboratories (Biological Division), Langley Court, Beckenham, Kent. Received for publication October 1, 1952. IN work on the action of proteolytic enzymes on antitoxin it was shown (Pope, 1939a) that when normal horse serum was submitted to the action of pepsin there was a rapid digestion of more than 95 per cent of the initial protein. When the heat-denaturation process (Pope, 1939b) was applied to normal horse serum almost all the original protein was lost. On the basis of these results it would have been expected that the application of these methods would lead to a uniform purification of antitoxin regardless of the initial titre of the antitoxic serum employed, had the antitoxin been the only new pepsin-resistant protein produced as a result of hyperimmunization. The results, however, showed very clearly that this was not the case. Generally, horses that had immunized rapidly to high titre gave good results-that is, a product with a high value in terms of units ofantitoxin per gramme ofprotein. The sera from horses that had responded poorly to immunization gave, as a rule, low " purity figures " in the peptic concen- tration process. Some further light was thrown on this problem when Pope, Stevens, Caspary and Fenton (1951) reported that in addition to the specific antitoxin present in anti-diphtheria serum there was present also a series of antibodies to antigens present in C. diphtheriae culture filtrates, and moreover that these other anti- bodies behaved in the peptic process as did the antitoxin itself. Thus, the final product obtained after peptic concentration contained a series of antibodies in addition to the antitoxin of which only the antitoxin was determined quantita- tively. Pope et at. have indicated that at least 24 antibodies were present in some of the antitoxic sera which they examined. The presence of a large number of antigens in the culture filtrates from C. diphtheriae may account for the failure of some horses to give a good antitoxin response on immunization, since it is only the antitoxin that is determined and not the total production of antibodies of different kinds. For exaample, a horse showing a poor antitoxin response may have produced large amounts of one or several other antibodies. If this is, in fact, the explanation for the low unit per gramme protein figures for certain antitoxic sera after peptic concentration, then it should be possible to show that almost all the protein that has survived the. action of pepsin, followed by heat denaturation, has antibody properties of one kind or another. The most direct way of showing this would be by the precipitation (floccu- lation) of the non-antitoxic antibodies with their antigens, leaving in this way PURIFICATION OF ANTITOXIN 57 the specific antitoxin as the only remaining immunologically reactive protein. This, of course, requires the availability of all these antigens free from specific toxin, and it is in this respect that we have met our greatest difficulties in this work. However, considerable progress has been made, and it is the object of thfs paper to show that there is much evidence to support the view that the low unit/g. protein value obtained after peptic concentration of antitoxic sera from horses showing a poor response on hyperimmunization is due to the presence of other antibody proteins diluting the true antitoxin. MATERIALS AND METHODS. Antitoxin. For most of the work reported here antitoxin concentrated by the peptic method (Pope, 1939b) was used; we selected a batch of material which had the very low purity figure of 16,000 units/g. protein. If it could be shown that the bulk of the protein present could be accounted for as antibodies other than antitoxin it would be clear proof of the view expressed previously. In addition we have used antitoxic serum derived from a horse specially immunized with diphtheria toxoid which had been submitted to heat treatment. It was reported by Pope et al. (1951) that crude culture filtrates from C. diphtheriae contained a large number of proteolytic enzymes (about 17), and that these were heat-labile. When diphtheria toxoid was heated rapidly to a temperature of 900, held at this temperature for a few seconds and then rapidly cooled, it was found that the proteolytic enzymes were inactivated and other antigens had their antigenic properties impaired. There was no loss of Lf in the in vitro test, indicating no loss of combining value with antitoxin, and no obvious change in the time of flocculation (Kf). However, the use of " flash-heated "toxoid for the hyper-immunization of the horse resulted in the production of serum giving far fewer lines in the diffusion test reported by Pope et al. (1951), indicating the production of a smaller number of antibodies than usual. This serum (MS 381) was studied both after concentration by ammonium sulphate (30-S50 per cent saturation fraction) and peptic concentration (Pope, 1939b). Other batches of peptic antitoxin were used on a smaller scale in order to confirm certain points. All the antitoxins used in this work were assayed by both in vivo and in vitro methods and the "Lr/Lf ratio " was substantially 1-0. This was also true for absorbed antitoxins which were tested similarly. Both tests were related to the International Standards through our laboratory sub-standards. Antigen Sources. Three types of products containing antigens have been used in this work: (a) Culture filtrates from C. diphtheriae grown in the presence of excess iron to inhibit toxin production. (b) Suspensions of C. diphtheriae in 50 per cent diethylene glycol. (c) Culture filtrates from C. diphtheriae in which the specific toxin has been destroyed. These are described more fully below. Excesm iron culture filtrates. The effect of a high iron content in culture medium used for the production of diphtheria toxin was reported by Pope (1932), who showed that although growth of C. diphtheriae was quite good on iron-rich medium, the production of toxin was almost inhibited. Later, Pappenheimer and Johnson (1937), working with a medium of different composition, extended our knowledge of the r6le of iron in the production of toxin, and also showed that in the presence of 0-5 mg. iron/litre of medium, toxin production was practically inhibited. Culture filtrates from C. diphtheriae grown in the presence of excess iron (4-0 mg. Fe... /litre) using tryptic digest medium (Linggood and Fenton, 1947) have been employed in this work. The amount of iron required to inhibit toxin production depends on the composition of the medium used. These culture filtrates were concentrated by ultrafiltration using ultrafilters of the type described by Harms (1948). Based on the reduction in volume, a normal toxic .58 C. G. POPE AND MURIEL F. STEVENS culture filtrate with an original value of about 80 Lf/ml. would have had a final value of about 4000 Lf/ml. The iron-filtrate concentrates had a value of 0-4 Lf/ml. (determined by blend test (Glenny and Okell, 1924)) on the concentrates. It is clear, therefore, that even after concentration these excess-iron culture filtrates are substantially free from specific toxin. It was hoped that these culture filtrates would provide a complete source of antigens, other than the specific toxin, but, as will be shown in this paper, this proved not to be true. Antigens from C. diphtheriae. We used the collected C. diphtheriae available from the large scale production of toxin or from cultures from excess-iron medium. The well-washed bacteria were pressed until fairly dry and then suspended to form a thick cream in 50 per cent diethylene glycol/water. Morgan (1937) used anhydrous diethylene glycol for the extraction of toxic 0 antigenic complexes from the smooth strains of Sh. shizae. The extraction of dried C. diphtheriae with anhydrous diethylene glycol appeared to be less effective than the use of 50 per cent diethylene glycol. These suspensions were readily agglutinated by crude peptic antitoxins, and could be used for absorption of some of the antibodies present. Other antigen preparations were obtained from these bacterial suspensions, for example, by digestion with trypsin or papain. The soluble products were tested for antigens or haptens by the diffusion method described by Pope et al. (1951) and by the optical method described later in this paper. Only a portion of the total antigen system was found in these prepara- tions. Antigens derived from C. diphtheriae crude toxin filtrates. In the course of this work we made many attempts to find a method whereby the specific toxin could be destroyed without the destruction of the other antigens present in the crude toxic filtrate. We were not completely successful in achieving this, but through the use of pepsin at controlled pH values we were able to destroy almost all the toxin, and yet leave many of the antigens in a form where they flocculated readily with crude peptic antitoxin. There is, however, evidence that some of the antigens present resembled the specific toxin in that they too were destroyed by pepsin. For most work we found it preferable to use ultrafiltered toxin, diluted to 500 Lf/ml. and adjusted to pH 4-7-5 0, the digestion being carried out at 200 for several days. The amount of pepsin was not critical, and we used 0-5 g./litre of 1/3000 B.P. strength pepsin. Small samples, neutralized to pH 8-855, were tested at intervals using the blend test (Glenny and Okell, 1924) until the value of the specific toxin had fallen to less than 4 units/ml.