pH effects accompanying serological reactions
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Contributions of a number of scientists to the study of the operating forces in serological reactions led to the theory that at least some ionized groups must be involved in the specific combination of the antibody and antigen molecules. This theory, summarized by Pauling et al. (60), states that the affinity for specific antibody-antigen union depends on ordinary " short range" forces. One of these forces, Coulomb attraction, operates between oppositely charged ionized groups arranged in complementary pattern on the molecules with the serological properties. The advancement of ionized groups participation theory raised the question of whether there is a change in hydrogen ion concentration following the serological reactions. Hirsh (40) and later Smith ana Marrack (71) tried to solve the problem by determining the pH in diphtheria toxoid and its anti toxin reactions. Whereas Hirsh found great changes in pH during the serological reactions, Smith and Marrack reported that they could not find any pH change. In an effort to add some clarification to this problem this work was designed to determine the pH following the reactions of two serological systems: 1. Plant hemagglutinin (lectin) plus blood group A substance. 2. Diphtheria toxoid plus diphtheria antitoxin. Those systems were preferentially selected because the high percentage of lectins and diphtheria antitoxin are specifically precipitable with their corresponding antigens. Therefore they contain less inactive proteins to obscure any pH changes which may result from the union of antigen and antibody. The solutions of lectins and A substance were obtained from Dr. W. C. Boyd's laboratory at Boston University School of Medicine. The procedure of preparation is described by Boyd et al. (14). The A substance was prepared from commercial hog gastric mucin, essentially by the 90% phenol method of Morgan (1). Th e diphtheria toxoid and antitoxin were obtained from the Vaccine Department of Massachusetts Public Health Biologic Laboratories, and were prepared essentially by the methods of Banzhaf (3) and Mueller (54). The following an analytical data of those substances were reported by the suppliers: 1. Lectin, 40% concentr. in saline soln., 1.68 mg N/ml, 32-36% specifically precipitable antigens and antibodies 2. A substance, 1% concentr. in saline soln., 33% specifically precipitable antigens and antibodies 3. Diptheria antitoxin, 4200 Standard Units concentration, 6.48 mg N/ml, 29% (calculated) specifically precipitable antigens and antibodies 4. Diptheria toxoid, 4200 Lf Units concentration, 1.85 mg N/ml The pH's of the above solutions were measured, then the antibodies were mixed with their corresponding antigens by equal volumes and the pH's of the reacting mixtures were determined: a. Right after mixing b. 15 minutes after mixing c. 24 hours (refrigerated) after mixing. Using the weak solutions of HCl and HaOH the solutions of lectin and A substance were adjusted to the following pH's: 3.00; 3.70; 4.50; 7.00. By the same method the solutions of diphtheria toxoid end antitoxin were brought to the pH's: 4.00; 4.80; 5.83; 6.70; 7.50. Then equal volumes of the solutions of lectin and A substance were mixed together end the hydrogen ion concentration of the mixture was determined: a. Right after mixing b. 15 minutes after mixing c. 24 hours after mixing (refrigerated). Employing the same technique the solutions of diphtheria toxoid and antitoxin were combined by equal volumes and the pH readings were taken. In order to obtain some information about the buffer capacity of lectin, diphtheria antitoxin and A substance, 25 ml of each of those solutions was titrated with 0.1 N HCl and NaOH, and pH readings were taken after the addition of every 0.5 ml of acid or base. The instrument used fur pH determination was Cambridge Instrument Company pH meter No. 0-181356. The efficiency of the apparatus was found to be 0.03 pH units. The glassware used was dry and free of contamination. In the case of toxoid and antitoxin, the sterility was preserved throughout the work. RESULTS In a series of measurements performed at a wide range of pydrogen ion concentration of the reactants, no pH change was found in those serological reactions. The pH of combined solutions of lectin and A substance, as well as diphtheria toxoid and antitoxin, remained the same as the pH of those solutions before mixing. At pH 3.00 combined lectih and A substance solutions showed no sign of reaction; even 24 hours later no precipitate appeared in the test tube. The rest of the solutions, when combined at their corresponding pH, showed normal preciptin reaction. The time required for the precipitation was not determined. The buffer capacity of lectin and diphtheria antitoxin (computed from titration curves) was found to be 4.65 x 10^-5 Eqv/pH and 1.58 x 10^-4 Eqv/pH respectively. The titration curves also disclosed that,at this buffer power of the solutions and pH meter sensitivity of 0.03 pH units, the hydrogen ion produced or neutralized must be at least 1.14 Eqv/mole of active lectin and 2.66 Eqv/mole of active diphtheria antitoxin so that pH changes could be detected in those serological solutions. SUMMARY The pH following the reaction of two serological systems a. Plant hemagglutinin (lectin) and blood group A substance b. Diphtheria toxoid and diphtheria antitoxin was determined. No detectable change of pH was found on comparing the pH of the starting solutions with the pH found after mixing these serological solutions.
Thesis (M.A.)--Boston University