STUDIES ON THE NATURE OF ANTIBODIES PRODUCED IN VITRO FROM BACTERIA WITH HYDROGEN PEROXIDE AND HEAT EDWARD C, ROSENOW From the Gates and Crellin Laboratories of Chemistry, California Institute of Technology, Pasadena, California* Received for publication July 5, 1946 Degradation and agglutination of organisms, destruction of toxic components and production of “agglutinins” and “precipitins” occurred when gram-positive bacteria in NaCl solution-suspensions were subjected to prolonged heating in the autoclave (1). Ina further study it was found that much less heat was required if an oxidizing agent, such as H,Oz, was added to suspensions of both gram- positive and gram-negative bacteria to produce substances resembling “natural” agglutinins and precipitins in high titer (2,3). The thermal “antibodies” thus obtained were specific but hed greater avidity for closely related organisms and their polysaccharides than corresponding natural antibodies ‘They had pro- tective action in vivo and in agreement with natural antibodies had little or no bactericidal action in vitro. They were highly stable when kept in the acid range of pH, the reaction essential for their ready production, moderately stable at neutrality and highly labile when alkalinized. When added to protei solutions, at room-temperature, agglutinin-titers usually diminished sharply. Specific agglutinins were the lest to disappear (2, 3). ‘The antibodies prepared with heat alone had extreme avidity for particulate matter and were not filtrable (1). Those prepared with H,O; and heat passed through fritted glass, diatomaceous earth and Seitz filters with little or no loss of titer. They were slowly dialyzable through cellophane against running water and running isotonic solutions of sodium chloride, were distillable on boiling, and were demonstrable in high titer in solutions of the dry residue (2,3). Erythem- atous cutaneous antibody-antigen reactions following intradermal injection of thermal antibodies paralleled the reactions obtained with the corresponding natural antibodies prepared respectively in vitro and in the horse from strepto- cocci isolated in studies of various diseases due to or associated with alpha strep- tococei. Experiments designed to determine more precisely the nature of thermal anti- bodies and their relation to natural antibodies comprise the basis of this report. METHODS Since natural antibodies are attached to, or are an integral part of, the globu- lin-fraction of the scrum of animals after recovery from natural and experimental infections and after immunization, attempts were made to produce antibodies in vitro from solutions of globulin adjusted to pH 6.5 without and with bacteria, * This work was aided by a grant for Medical Research from Mrs. Hobart Johnson of Madison, Wisconsin. Grateful acknowledgment is hereby made. 219220 EDWARD C. ROSENOW with H,0,, heat, and hydrostatic pressure, and since the “antibodies” produced from bacteria in NaCl solution were diffusible, differing in this respect, from “natural” antibodies attempts were made to “conjugate” thermal aggli- tinins onto gamma human globulin, human serum and other protein-solu- tions. We are indebted to Dan Campbell for a highly purified gamma human globulin used in these experiments which was prepared by E. J. Cohn. Ac- cordingly, solutions containing high titers of thermal agglutinins were mixed with globulin and other protein solutions of different strengths and the mixtures subjected to 65 C for 10 or 20 minutes and then to 10,000 pounds hydrostatic pressure per square inch for 10 minutes. The effect on agglutinin-titer, dialyza- bility through cellophane, distillsbility on boiling and other properties of such treated mixtures were studied extensively. ‘The temperature of 65 C is about the highest to which proteins can be heated at the pH employed, without causing very rapid denaturation. Pressures of the magnitude used have been found to influence markedly the rate of certain re- actions involving proteins, eg., they greatly retard the denaturation of serum globulin at 65 C at a pH near neutrality (4) indicating that the reaction proceeds with a large molecular volume increase of activation (5-7). ‘These pressures may likewise cause certain reversibly denatured, inactive enzyme-systems to become active again, by counteracting the volume increase of reaction in the equilibrium concemed (7). Thus, it was reasonable to suppose that pressure might influence the rate of the reaction between the protein and thermal antibody. The results indicate that such was the case, for the titer of pressurized specimens was always significantly higher than that of the unpressured protein-plus-antibody speci- mens, similarly treated with heat but at normal pressure. It is evident that the reaction in question is accelerated by pressure and hence proceeds with a volume- decrease of activation or reaction. The methods used in this study for the isolation and preservation of specificity of streptococei and pneumococci, for the production of antibodies or antibody- like substances in vitro, for determining the agglutinin and precipitin-titers and for dialyzing and distilling the in vitro produced antibodies were essentially similar to those employed in previous studies (2, 3, 8). ‘The solutions of anti- bodies used were prepared unless otherwise indicated by diluting the dense sus- pensions containing 10" organisms per ml in glycerol 2 parts and 25 per cent solution of NaCl 1 part, 100-fold with isotonic solution of NaCl or protein-solu- tions, adding 1.5 per cent H,O; and autoclaving for 1 hour at 17 pounds pressure. ‘The agglutinative tests were made by placing 0.2 ml of five-fold dilutions (1-10 to 1-1,250; 1-20 to 1-2,500) or 1-100 to 1-12,500 of “antibody” made in NaCl solution containing 0.2 per cent phenol into test tubes (3” x 3”) and adding to these 0.2 ml of bacterial suspensions containing approximately 5 X 10° organ- isms per ml of NaCl solution also containing 0.2 per cent phenol and 0.125 per cent gelatin. These suspensions were kept at room temperature for from 4 to 6 1 Tam indebted to Professors Linus Pauling and Dan Campbell of the California Institute of technology and to Professor Frank H. Johnson of Princeton University for their coopers tion and suggestions.ANTIBODIES PRODUCED WITH HYDROGEN PEROXIDE AND HEAT 221 hours to allow the clumped organisms to settle, or they were lightly centrifuged, and the supernatant evenly dispersed suspensions were used. ‘The small amount of gelatin prevented “spontaneous” agglutination. The mixtures were thor- oughly shaken and placed in the incubator at 48 to 50 C for 18 to 24 hours (8), when readings were made, In order that the degree of total agglutination of the different strains by the four dilutions of the different materials containing agglu- tinin can be readily visualized in graphs and tables and for the sake of brevity, the percentages of total possible agglutination are given. The maximal or 4 plus agglutination in each of the 4 dilutions, or a total of 16/16 would be 100 per cent; a total of 5 for the 4 dilutions would be x or 31 per cent, ete. Interfacial precipitation-tests were made in small tubes (5 x 25 mm) by super- mposing the solutions of specific polysaccharide undiluted and diluted 1-5 and Feroontage of total, ‘poaaible agglutination at Fivye-folé @ilutions of 1-20 to 1-2, 500 tcc fy Beterelogens strep ‘tococcus SS8ssssss oo Materials itn Buttered Rach solution, 65 70,000, 000,000 por ml, fren Genae glycerine NaC solution suspension, Fig. 1. Propuction or THERMAL AGGLUTININS FROM GLOBULIN AND FROM STREPTOcoccT SusrenvEp 1x GLOBULIN AnD IN NaCl Souutions wire H;0; anv Hear 1-10 onto undiluted antibody. ‘The undiluted solutions of polysaccharides pre- pared by the Lancefield method contained the polysaccharide from 2 X 10" or 10" organisms per ml. The degree of clouding or precipitation at the interface after 30 minutes at room-temperature and 18 hours at 10 C, respectively, was recorded according to the scale of 0 to 4 plus. ‘RESULTS OF EXPERIMENTS ON PRODUCTION OF ANTIBODIES It was found (fig. 1) that agglutinins were not formed from globulin at 23 C without and with H,0, but were formed in low titer with H,0; at 123 C for 1 hour in the autoclave and in high titer with H,0, at 123 C for 1 hour after adding 10,000,000,000 bacteria per ml. Agglutinins were not formed at 23 C nor 123 C from buffered NaCI solution (pH 6.5) after adding H,O, but were formed in moderate titer on adding bacteria, H,0; and autoclaving for 1 hour. The effect of 10,000 pounds hydrostatic pressure without and with heat on the production of agglutinins from streptococci suspended in NaCl solution was also222 EDWARD C. ROSENOW studied. A suspension in NaCl solution of (alpha streptococci, 10,000,000,000 per ml representing a 1-100 dilution of the dense glycerine-NaC! solution sus- pension was divided into 6 equal parts after adding 1.5 per cent Has. Part I was heated at 65 C under 10,000 Ib pressure for 45 hours. Part 2 was heated at 65 C at atmospheric pressure for 45 hours. Part 3 was kept at room- temperature (23 C) at atmospheric pressure for 45 hours. Part 4 was kept at room-temperature (23 C) under atmospheric pressure for 44 hours, and then autoclaved at 17 Ib (123 C) pressure for 1 hour. Part 5 was kept at 10 C for 45 hours, and Part 6 at 10 C for 44 hours and then autoclaved at 17 Ib pressure for 1 hour. The agglutinin-titers for the homologous and heterologous streptococcus and for Staphylococcus aureus were roughly proportional to the heat applied but bore little or no relation to pressure. AGGLUTININ-TITER OF THERMAL ANTIBODIES BEFORE AND AFTER PROCEDURES TO CONJUGATE ONTO GLOBULIN, ALBUMIN, AND SERUM Studies on the effect of heat (65 C for 10 or 20 min) and of hydrostatic pressure 10,000 lb per square inch for 10 minutes on the agglutinin-titer of thermal anti- bodies after adding them to solutions of purified human globulin or egg-albumin of different strengths were studied in 6 different experiments. One part of thermal antibody was routinely added to 4 parts of the respective solutions of globulin or albumin and as a control 1 part antibody to 4 parts of the buffered NaCl solution. ‘The percentage of antibody in dilution of 1-100 to 1-12,500 was determined for the homologous strains of streptococcus, for pneumococci, for Staphylococcus aureus or E. coli. The effect of heat and pressure on the agglutinin-titer of mix- tures containing s constant amount of thermal antibody and globulin-solutions of different strengths were first studied. The agglutinin-titer for the homologous streptococcus in one experiment applying heat and pressure to mixtures con- taining 1 part of thermal streptococcal antibody plus 4 parts respectively of 1, 0.5, 0.25, 0.125 and 0.06 per cent solutions of globulin in phosphate-buffered (pH 6.5) NaCI solution and as a control 1 part of antibody to the buffered NaCl solution without globulin were 88, 81, 75, 69, 56 and 56 per cent respectively. In this as in other experiments subjecting 1-5 dilutions of antibody in NaCl solutions to heat and pressure caused no demonstrable change in agglutinin-titer over unheated mixtures. Adding antibody-solutions to globulin or albumin and not heating usually caused a sharp drop in agglutinin-titer, sometimes no change, and only rarely an increase in titer whereas subjecting 1-5 dilutions of antibody in globulin- or albumin-solutions to hest and pressure consistently caused an in- crease in agglutinin-titer. The results in the six different experiments were essentially similar to those obtained in the experiment summarized in figure 2. The agglutinin-titers of the 1-5 dilution of streptococcal antibody in buffered NaCl solution used in the experiments summarized in figure 2 that were not heated and not subjected to pressure were not changed while that of the pneumo- coccic antibody was increased wheress when the streptococcie and pneumo- cocci antibodies were mixed with globulin-solution and heated to 65 C for 20 minutes and cooled slowly under atmospheric pressure agglutinin-titer in allANTIBODIES PRODUCED WITH HYDROGEN PEROXIDE AND HEAT 223 instances increased moderately. When heated at 65 C for 20 minutes and then subjected to 10,000 Ib pressure for 10 minutes and cooled slowly the agglutinin titer was greatly increased in all instances. This was greater in mixtures con- taining 0.4 per cent than 0.2 per cent concentrations of globulin. ‘The increase in agglutination after conjugation of antibody onto globulin was especially grest or specific for the respective homologous strains of streptococci and pneumo- cocci (fig. 2). The percentage of agglutination obtained in experiments not shown in fig. 2 ranged from 25 to 44 per cent and the endpoint at which agglu- tination occurred ranged from 1-500 to 1-2,500 for the control unconjugated or unheated solutions while the percentage of agglutination of the corresponding solutions conjugated onto globulin with heat and pressure ranged from 70 to 94 per cent and the endpoint of agglutination ranged from 112,500 to 1-62,500 and [easter er ERE EY SE BY 7 bartered bo GS. WEED pos Fra. 2. Consucarion or Ta=nMat SrmePTococcaL AND PNEUMOCOCCAL AGGLUTININS ONTO ‘Human Giosuuin sy Hzat axp Hypnostatic Pazssune in one experiment significant agglutination of the homologous suspension of pneumococcus occurred at dilutions up to 1-312,500. The detailed results and striking specificity on high dilution of thermal antibody in this experiment are depicted in table 1, In still another experiment made with a solution of strepto- coccal antibody similar specific agglutination of the homologous streptococcus occurred at progressive five-fold dilutions up to 1-312,500. Numerous experiments were made in attempts to conjugate the “raw” thermal agglutinins produced from suspensions or extracts of organisms in isotonic solu- tions of sodium chloride with H,O; and heat onto gamma human globulin and human serum-albumin, human, rabbit, and horse serum, onto solutions of gela- tin, protein-hydrolysates and acacia with heat (65 C) alone at atmospheric pres- sure. The results in one experiment with human serum are shown graphically in fig. 3. The complete absence of agglutination in 10 per cont human serum plus one part NaC! solution after heating, the low agglutinin titer of human serum plus raw agglutinin before heating and the striking increase in titer after heating and the remarkably high titer of agglutination through three successive 1-5 dilu- tions of the conjugated agglutinins are strikingly shown.224 EDWARD C. ROSENOW TABLE 1 Tnerease in agglutinin-titer of thermal antibody when conjugated ‘onto globulin by heat and pressure screen 9¢ Pa Eat ae ee Suet ace or ee az. |= noe Staphy Hocdecta | B. colt Suspension in 0.85% | 0.4% purified 1-100 |3 2 0 NaCl solution of | human globulin 1-500 | IS (21%)} 0 10,000,000,000pneu-| heated st 65°C. 1-2,500 [4 lo 0 mococei I, If, HIT} for 10min. then | 112,500 [3 4%)|0 ° a from dense | 10,000 Ibs. pres-| _ 1-62,500 |2 lo 0 glycerin NaCl solu-| sure for 10 min.| 1-312,500 2 (75%) lo 0 jo jo 0 tion suspension | and cooledelowly| 1-1, 562,500 autoclaved for 1 -————}—__| hour after adding | NaCl solution 1100 ls l4 (13%)] 0 1.5% H.Os. Super-/ heated at 65°C. 1-500 |4 lo 0 natant (pH 3.0) 1| 10 min. then at 1-2, 500 |3 (38%)|0 0 part + 4 parts 10,000 Ibs. pres-| 1-12, 500 [2 (54%)| lo 0 sure 10min. and | —_ 1-62,500 0 lo o cooled slowly 1-812, 500 |o lo 0 1-1, 562,500 |o lo 0 By Percentage of total possible agglutination ‘at five-fold dilutions 70] of 16100 to 1912,500 Blinte strestocoset 50 Ly emeseret types ho) LI Ure 59 [Staphylococous 20) ‘aureus 10 * 65° co 20 nimites atmospherio pressures Cooled slowly. o» Prepared fros HAC? solution suspension poeunccooct Types T, 11, HIT EF, 10,000,000, 000'per ale + 165% Hop, autoclaved 2 houre or Fic. 3, Consuaation or Raw Pxevmococcus AGGLUTININ ONTO HuMAN SeRUMANTIBODIES PRODUCED WITH HYDROGEN PEROXIDE AND HEAT 225 The agglutinin titer of 1-5 dilutions of a type 1 pneumococcal and an alpha streptococeal antibody in NaCl solution, in I per cent human serum-globulin and albumin, and in 10 per cent dilutions of human and rabbit serum respectively un- heated and heated to 65 C for 20 minutes and cooled slowly are shown graphically in fig. 4. It will be seen that the agglutinin titer of the dilutions with NaC! solution unheated and heated were moderately high and comparable. Agglutinin-titer of dilutions in globulin, albumin, and serums unheated usually were somewhat lower than titers in dilutions in NaCl solution and uniformly much lower than in the same dilutions in globulin, albumin, and serum after heating to 65 C and cooled slowly. ewe netagess te 2 soateestead (ea rapurimeoms |i ‘Souror ass treatuest of erm antidoey Sa Fic.4. AGGLurinin TITERS OF PNEUMOCOCCAL AND ALPHA STREPTococcAL THERMAL ANTIBODY BEFORE AND AFTER CONJUGATION ONTO Gamma GLOBULIN, Serum Atpran anp Nonmar Human anp Rassit Senum In these as in other experiments specificity was more pronounced after thermal antibodies were conjugated onto globulin, albumin, or serum than in the raw state and especially than identical dilutions in globulin, albumin, or serum that had not been heated. In altogether 13 instances the percentage of agglutination greater for the homologous organisms than for the most closely related heter- ologous organisms, or specific agglutination, averaged 4 per cent for the unheated mixtures of antibody and globulin, albumin, or serum whereas for the corres- ponding mixtures that had been heated averaged 21 per cent and in four experi- ments mixtures of antibody and globulin or albumin that had been heated and subjected to 10,000 Ib pressure the agglutination for the homologous strain averaged 49 per cent greater than for the heterologous strain. RESULTS ON DIALYSIE The effect of dialysis through cellophane against equivalent amount of NaCl solution, against running NaC! solution and running water on the agglutinin-226 EDWARD C, ROSENOW titer of streptococcal and pneumococcal thermal agglutinins was studied in parallel manner before and after conjugating them onto globulin. ‘The results of one dialyzing experiment against running NaCl solution are summarized in fig. 5. ‘The raw or unconjugated solutions and the conjugated solutions were placed in 10 ml amounts in cellophane bags 2.em in diameter and were dialyzed in separate beakers of 200 ml capacity. The NaCl solution from a 5-gallon bottle was made to flow into each beaker at the same rate. The NaCl solution sur- rounding the cellophane bags in the beakers was allowed to over-flow. The experiment was done in a cold room at a temperature of 15 C and the level of the solution inside of the bag was kept the same as the surrounding running NaCl solution in the beakers. The thread-tied seal at the bottom of the cellophane bag and defects in the cellophane were checked for air-tightness before and after Percent poseible agrlutinas Sion at five-fold adlutlons of 1-100 ‘to 112,500 Honolopous ‘ttreptcooeods Heterologous streptoscsous ‘stertylovocous Ssyseyss ss ‘¢ Propered from MaCl solution euspension ef alphn streptoceest, 10,000,000,000 per ale + 19K 1gdgy autoolayed ona boure ‘ov pattered to pil 6.5-with phosphate buffers Fro. 5. Aaonurtnaine Tiree axp DiaLysis oF THERMAL AGGLUTININ AGAINST RUNNING ‘NaCl SOLUTION BEFORE AND AFTER CoNsUGATION onTO HumAN Gamma GLOBULIN the experiment. The agglutinin-titer for the homologous and the heterologous strain of alpha streptococcus and Staphylococcus aureus was determined of the specimen within the cellophane bag and of the surrounding running NaC! solu- tion in the beaker after dialysis for 1}, 3 and 7 days respectively. During the 7 days, 38 liters of the running NaCl solution were used for each beaker. It will be seen that the agglutinin-titer of the unconjugated or “raw” antibody solution on dialysis diminished earlier and very much more than that of the con- jugated solution. Comparable results were obtained in three other similar ex- periments with human gamma globulin and with one each of a solution of purified serum-albumin (human) and a solution of egg-white when dialyzed against running NaCl solution at 6 C for 6 or 8 days, respectively. In all instances thermal agglutinins were rendered much less dislizable or nondialyzable through cellophane against running NaCI solutions after conjugation onto globulin orANTIBODIES PRODUCED WITH HYDROGEN PEROXIDE AND HEAT 227 albumin, Only traces of agglutinins were found in the running NaCl solution surrounding the cellophane bag in the beaker representing a possible dilution of antibody of 1-8,800, Agglutination was always less in the exposed running NaCl solution than in 1-12,500 dilutions of the dialysate. ‘The agglutinins in the “raw” state in NaCI solution were slowly dialyzable through cellophane against running NaCl solution, while those in solutions that had been added to serum were no longer or were much less dialyzable regardless TABLE 2 Agglutinating titer of thermal antibody before and after conjugation onto human serum and before and after dialysis against running NaCl eolution PERCENTAGE OF TOTAL FOSSIMLE AGGLUTINATION OF ‘moMoL0c0US AND HFTEEOLOCOUS ORGUOSMS Ar PIVE-YOLD DILU- "SIONS OF 1-100 To 1-12,900 OF RESPECLIVE ANTIBODY SOLUTIONS sourmon or emopy 4x0 mazar net akipat Palio heags eee ee One part | 10%human| Not heated) 31 18 13 13 ey 7 pneumo- | serum ‘coccus Type 1 Heated at| 44 | 44 | 50 “ “ 30 antibody °C. for +4parte| NaCleolu-| 20 min.| 25 | 31 | a1 0 9 o tion Cooled slowly One part | 10%human| Not heated) 18 | 44 | 31 oy “ 38 alpha serum strefto- coceus Heated at| 50 | 69 | 50 | 50 | 63 | 50 antibody 65° C. for! +4 parts | NaCleolu-| 20 min.| 31 | 38 | 31 o | 3% ° tion Cooled slowly whether heated or not. ‘The results in one such experiment are summarized in table 2. In other words, normal serum after conjugation with thermal antibody became similar to immune serum in agglutinating titer (fig. 2, table 2) and in nondislyzability (fig. 5). ‘The results on. dialysis through cellophane against equal amounts of NaCl solution for 6 days at 10 C showed that while agglutinins passed through the cellophane, there was little or no diminution of titer within the bag over that of the undialized control. Sometimes the agglutinin-titer in the NaCl solution surrounding the bag was only slightly less and at times as great but never greater than that within the bag. The most striking increase in agglutinin titer on prolonged dialysis at 10 C against equal amounts of NaCl solution both within228 EDWARD C. ROSENOW and surrounding the bag occurred in mixtures of thermal antibody and protein hydrolysates, amino acids and .2 per cent globulin which had not been heated. ‘This increase in total agglutinin-titer occutred in 6 other experiments with solutions containing agglutinins in especially high titer after prolonged dialysis against equal amounts of NaCl solution. Increase of agglutinins may have occurred during prolonged dialysis against running NaCl solution but this was not demonstrable due to excessive dilution. In three experiments dialysis was done simultaneously with the unconjugated thermal agglutinin against equal amounts of NaCl solution and against running NaCl solution. Slight or no diminution or actual increase occurred as equilibirum of agglutinin was ap- proached under the former conditions, while striking diminution occurred under the latter conditions in each experiment. ‘The agglutinin-titer of unconjugated thermal antibody disappeared in each of 4 experiments after prolonged dialysis against running water in which the reac- tion became neutral, pH 7.0, and in which heavy precipitation in the specimens occurred. When the acidity of the dialyzed, precipitated solutions was brought to the original acidity, pH 2.5, much of the precipitate went into solution and high although less than the original, agglutinin-titer returned. Control NaC] solution brought to pH 2.5 caused only slight agglutination in the 1-20 dilution, RESULTS ON DISTILLATION In previous studies it was found that thermal agglutinins and precipitins produced from bacteria suspended in solutions of sodium chloride and in solu- tions of protein-hydrolysates when heated with H,O: were not only dialyzable but distillable on boiling and were present in relatively high titer in solutions of the dry residue. Studies were therefore made to determine whether thermal agglutinins after conjugation onto globulin, albumin or serums were distillable. The procedures used and the amounts distilled were identical to those in the distillation of the “raw” agglutinins. Five or 10 ml of the conjugated solutions of antibody were placed in a Claisen distilling flask whose bulb was of 75 ml capacity. The de- livery tube to the condenser was 15 cm above the level of the fluid in the flask and the side arm from which the condensing tube extended was filled with fragments of glass and small glass balls. The bottom of the bulb containing the conjugated. antibody plus glass beads rested on an asbestos screen. Heat from a Bunsen flame was applied and the solution was boiled slowly to dryness. Carrying over of bubbles and visible vapor throughout the process was avoided in so far as possible. The distillate was brought to isotonicity with NaCl and the dry residue was dissolved in distilled water in amounts equivalent to the original solution. The agglutinin-titer of both the distillate and solution of the dry residue was determined in the usual manner. ‘Agglutinins were present in fairly high titer in the distillates of the “raw” pneumococcal and streptococcal antibody-solutions whereas after agglutinins were conjugated onto human gamma globulin, serum-albumin and normal human serum they resisted distillation completely while those conjugated onto rabbit- serum were carried over in low titer.ANTIBODIES PRODUCED WITH HYDROGEN PEROXIDE AND HEAT — 229 Evaporation to dryness of the pneumococcal antibody-solution previously conjugated onto human globulin, albumin, and human serum destroyed com- pletely the agglutinins effective for streptococci and staphylococci and destroyed ‘only part of the agglutinins specific for pneumococci. Whereas agglutinins for both pneumococci and streptococci were present in fair titer in solutions of the dry residue of the pneumococcal antibody conjugated onto rabbit-serum. Ag- glutinins for streptococci and pneumococci were present in low titer in solutions of the dry residues in case of streptococcal antibody conjugated onto human globulin, albumin, and serum and in moderate titer when conjugated onto rab- bit-serum. In order to check the possibility of droplet contamination of distillate by the method used additional experiments on distillation of antibody were done in which four fractionating, inverted, pear-shaped glass bulbs, creating a mul- tiple baffling effect were used. The solutions were boiled slowly with glass beads to avoid bumping, over a Bunsen flame in a 500 ml round bottom flask. The condensing tube outlet was 45 cm above the liquid level of 30 ml. The temperature of the room was 23 C. Visible spattering of droplets on the inside of the flask during boiling could not be detected higher than 4 cm above the level of the liquid. Agglutinin titer of distillate rendered isotonic and of solu~ tions of the residue in distilled water were comparable to those obtained with the Claisen distilling flask. SUMMARY AND COMMENTS The production of “agglutinins” from globulin with and without bacteria, the effect of hydrostatic pressure on agglutinin-production and the effect. on agglutinin-titer of antibodies produced tn vitro from baeterial in NaCl suspension with H,0; and heat when conjugated onto globulin, albumin, and serum with heat and pressure and with heat alone are reported. ‘Treatment of solutions of globulin with HO; and heat without bacteria re-~ sulted in formation of agglutinin in moderate titer while similar treatment: of suspensions of streptococei in solution of globulin gave high titers roughly pro- portional to the heat applied. Hydrostatic pressure at ordinary temperatures had no demonstrable effect on antibody-produetion. The destruction of pytogens in water and in solutions of gelatin with H.O: and heat reported by Campbell and Cherkin (9) are in accord with our experi- ments on the destruction of toxic components of bacteria and as this occurred the production of antibody from bacteria, from bacteria-free extracts, filtrates, dialysates and specific polysaccharides (2, 3). Diluting saline solutions of the in vitro produced antibodies with NaCI solu- tion reduced agelutinin-titer proportional to the dilution regardless of whether heated or not. When diluted similarly with solutions of globulin, albumin, or serum and not heated, agglutinin-titer was usually disproportionally greatly reduced and in sharp contrast when heated or conjugated onto globulin, albumin, or serum, agglutinin-titer, especially specific titer, was usually greatly increased and when heated and subjected to excessive hydrostatie pressure both total and specific titers were still further incressed.230 EDWARD C, ROSENOW Saline solutions of thermal antibodies whether heated or not were more di- alyzable through cellophane against running NaCl solution and more distillable on boiling than corresponding unheated or heated solutions of antibodies in globulin, albumin, or serum. Both unconjugated and conjugated solutions lost agglutinin-titers rapidly when dialyzed against running water. As this occurred precipitates appeared in the solutions dialyzed, especially in those containing globulin, albumin, or serum, and when brought to isotonicity with NaCl and the original acidity with HCI, partial restoration of agglutinin-titer occurred as part of the precipitate went into solution. Moreover, when both types of dilutions, especially those conjugated onto globulin or other protein-containing solutions were dialyzed at about 10 C against equal amounts of NaCl solution for from 6 to 8 days, agglutinin-titer often markedly increased in the specimens and high titers ap- peared in thé NaCl solution surrounding the dialyzing bag. ‘The increased titers were found not to be due to increased agglutinability of organisms in the suspensions. Corresponding solutions in glass containers kept under identical conditions except for the dialyzing membrane and the sur- rounding NaCl solution repeatedly tested in parallel, with the titer when kept at 10 C, but when kept at 37, 56, 65 or 72 C for from 1 to 12 weeks, increase in agglutinin-titer occurred, especially in specific titer, roughly proportional to time and temperature at which kept (2, 3). Boiling solutions of natural streptococcal and pneumococcal antibody and solutions of corresponding artificial antibody after conjugation onto human globulin, albumin, or serum to dryness caused somewhat greater reduction in agglutinin-titer of solutions of the dry residue than boiling to dryness the cor- responding raw unconjugated solutions of ‘‘antibody.”” It is not claimed that the experiments on distillation establish true volatility ‘of the raw and non-volatility of conjugated thermal antibody. However, they do show that artificial agglutinins in the raw state in NaCl solution are carried over on boiling in higher titer under identical conditions than after con- jugation onto globulin, albumin, or serum. ‘The important question whether the increase in agglutinin-titer of specimens of antibody within the cellophane bag and the dialysate obtained during di- alysis against equal amounts of NaCl solution, of distillate and solution of the dry residue after slow boiling to dryness, and the high titer of agglutinins on successive dilutions of conjugated agglutinin, represents formation or synthesis of agglutinins as would seem, or whether due to altered complex relationship between concentrations of free and conjugated protein, antibody, and bacterial cells in the agglutinin tests remains uncertain. ‘The diffusible agglutinins produced in vitro by the oxidation of bacterial antigen caused normal human globulin and serum to acquire high and specific agglutinin-titers or to become like antibody-globulin and antiserum respectively by subjecting mixtures to moderate heat or to heat and hydrostatic pressure. In previous studies (2, 3) it was found 1) that antibody formation with HO:ANTIBODIES PRODUCED WITH HYDROGEN PEROXIDE AND HEAT 231 and heat occurred over an extremely wide range in the number of organisms in suspensions, from 10" to 10° per ml. 2) That antibody-formation under oxida- tive reactions occurred earlier and in relatively higher titer in bacteria-free dialysates of suspensions and in solutions and filtrates of solutions of specific polysaccharides than from corresponding suspensions of the bacteria, 3) That the bacterial cell appeared to contain antagonistic or toxic substances which interfered with or were highly resistant to antibody-formation and which re- quired more prolonged exposure to oxidative reactions for conversion into sub- stances resembling antibody. 4) That subjecting solutions of globulin or protein-solutions to oxidative reactions with HO; and heat produced nonspecific agglutinins in relatively low titer in the absence of bacteria and specific ag- glutinins in high titer with bacteria and 5) that small concentrations of HO; at body-temperature sufficed for the formation of agglutinins and precipitins in fair titer in vitro. The data obtained on the in vitro production of antibodies by oxidative re- sctioas and their conjugation onto globulin indicate 1) that perhaps the in tivo destruction of antigen by reticuloendothelial cells, so strikingly shown by Sabin (10) may be due to enzymatic oxidation of antigen into diffusible antibody such as we have produced in vitro and 2) that diffusible antibody combines with normal globulin in vivo in lymphocytes (11) reticulonendothelial and other cells and in serum as it does tn vitro to form antibody-globulin rather than that antigen causes reticuloendothelial and other cells to acquire a new function namely that of synthesizing and secreting a modified serum-globulin, the anti- body-globulin (12). In the experiments with bacteria-free extracts, filtrates and dialysates minute amounts of bacterial antigen sufficed for the production of diffusible antibody which when conjugated onto normal globulin, caused normal globulin to ac- quire extremely high specific agglutinin titer (table 1) and other properties resembling antibody globulin. ‘The striking increase in agglutinin titer which occurred on prolonged di- alysis against equal amounts of NaCl solution of unheated mixtures of diffusible antibody and protein hydrolysates, amino acids and globulin respectively and the high agglutinin titer on successive dilution of diffusible antibody conjugated onto globulin furthermore indicate that under proper conditions extremely small amounts of diffusible antibody and hence antigen from which derived sufficed to cause normal globulin to take on properties resembling antibody globulin. All indicate that perhaps most of the weight of natural antibody globulin represents globulin and only a small fraction antibody from antecedent antigen. The results reported herewith are considered corroborative and as extensions of the original experiments on the én vitro production of antibodies (13, 14), and the studies on the tn titro production of substances resembling antibodies from bacteria without adding a sensitizing protein by prolonged exposure to heat in the autoclave (1). Results of studies on the chemical nature of the232 EDWARD C. ROSENOW substances resembling antibodies produced by oxidation and the protective action of the unconjugated and conjugated antibodies now in progress will be reported in due time. REFERENCES 1, Rosenow, B.C, 1945 The production in vitro of substances resembling antibodies from bacteria Inf. Dis., 76: 163-178 2. Rossow, E.C. In press The production in vitro from bacteria of substances re- ‘sembling “natural” agglutinins and precipitins. 3. Rossow, B.C. In press The production in vitro from protein and other solutions without and with bacteria of substances resembling ‘‘natural”’ agglutinins and pre- cipitins. 4, Jounson, F. H., ap Caurszut, D.H. 1945 Retardation of protein denaturation by hydrostatic pressure, J. Coll. Comp. Physiol., 28: 43-46 5. Evaro, H. 1935 Activated complex in chemical reactions, J. Chem. Physics, 8: 107- 118 6. Grasstons, 8., Laure, K. J. axo Ermine, H. 1941 The theory of rate processes. New York, McGraw-Hill. 7. Jounson, F. H., Eran, H., Sreetar, R., Cuaruay, H., Huer, C. ano Gurnanpr, G. 1945 The nature and control of reactions in bioluminescence. J. Gen. Physiol., 28: 463-537. 8. Rossnow, E. C. 1939 Studies on specificity of streptococci isolated in studies of + encephalitis, Arch. Int. Med., 64: 1197-1221. 9. Caupnnit, D, H., AND Cuenmin, A. 1945 The dextruction of pyrogens by hydrogen peroxide. Science, 102: 535-636. 10, Saniy, Frorence R. 1939 Cellular reactions to a dye-protein with a concept of the mechanism of antibody formation. J. Exp. Med., 70: 67-82. 11. Davonzarr, T. F., Cuasz, J. H., ano Warre, A. 1944 The determination of anti- bodies in lymphocytes. Proc. Soc. Exp. Biol. and Med., B7: 295-208. 12, Hetomuszncsn, M., ano Kanat, B.A. 1936 Quantitative studies on bacterial agglu- tination; quantitative theory of bacterial agglutination. Proc. Soc. Exp. Biol. and Med. , 36: 301-303. 13. Pacuivo,L., ano Cawppert,D.H. 192 Manufactureofantibodiesin vitro. J.Exp. Med., 76: 211-220. 14. Pauuina, L., Caursct, D, H., xp Pressman, D. 1943 Nature of forces between antigen and antibody and of precipitation reactions, Physiol. Rev. 29:203-219.