Studies on the Nature of Antibodies Produced in Vitro from Bacteria with Hydrogen Peroxide and Heat
Authors: Rosenow Edward C. 
Published: January 2023

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 in vitro from bacterial in NaCl suspension with H2O2 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 H2O2 and heat without bacteria resulted in formation of agglutinin in moderate titer while similar treatment of suspensions of streptococci in solution of globulin gave high titers roughly proportional to the heat applied. Hydrostatic pressure at ordinary temperatures had no demonstrable effect on antibody-production.
The destruction of pyrogens in water and in solutions of gelatin with H2O2 and heat reported by Campbell and Cherkin (9) are in accord with our experiments 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 NaCl solution reduced agglutinin-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 hydrostatic pressure both total and specific titers were still further increased.
Saline solutions of thermal antibodies whether heated or not were more dialyzable 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 HCl, 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 appeared in the 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 surrounding 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 corresponding 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 conjugation 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 dialysis 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 H2O2 and heat occurred over an extremely wide range in the number of organisms in suspensions, from 1011 to 103 per ml. 2) That antibody-formation under oxidative 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 required more prolonged exposure to oxidative reactions for conversion into substances resembling antibody. 4) That subjecting solutions of globulin or protein-solutions to oxidative reactions with H2O2 and heat produced nonspecific agglutinins in relatively low titer in the absence of bacteria and specific agglutinins in high titer with bacteria and 5) that small concentrations of H2O2 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 reactions and their conjugation onto globulin indicate 1) that perhaps the in vivo 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 in 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 antibody-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 acquire extremely high specific agglutinin titer (table 1) and other properties resembling antibody globulin.
The striking increase in agglutinin titer which occurred on prolonged dialysis 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 in vitro production of antibodies (13, 14), and the studies on the in vitro 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 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.