THE MOLECULAR WEIGHT OF ANTIBODIES

1. Highly purified preparations of homogeneous antibody can be made by the salt dissociation methods (6, 7) without any change in sedimentation due to the method of purification. 2. Antibodies prepared from sera of various animal species fall into two groups as regards molecular weight; in one group cow, horse, and pig, a heavy molecule of molecular weight 990,000 is formed; in human being, rabbit, and monkey, the molecular size is that of the normal γ serum globulin. Both types of antibody molecules are either not compact or not spherical since the frictional ratios f/f0 are 2.0 and 1.5 respectively. 3. Horse antibody shows an unchanged activity and sedimentation diagram between pH 3.44–9.06, although there is some aggregation at the more acid and some dissociation at the more alkaline pH. At pH 1.44 the antibody activity is unchanged but some breakdown of the molecule takes place. At pH 12.4 activity is destroyed and the molecule is completely broken down. 4. Some horse antibody preparations show evidence of breakdown of the antibody into inhomogeneous material on continued immunization over a long period

AN ELECTROPHORETIC STUDY OF IMMUNE SERA AND PURIFIED ANTIBODY PREPARATIONS

1. Antibody produced in the horse migrates as a new serum component between the β and γ components, whereas rabbit antibody is electrophoretically identical with the γ globulin component of the serum. 2. In rabbit and monkey antisera the percentage of antibody in the serum and in the γ globulin fraction can be determined by integration of the electrophoresis diagrams of unabsorbed and absorbed sera. Antibody solutions of high purity can be obtained by electrophoretic isolation of the γ globulin of rabbit antisera in which the percentage of antibody to total γ globulin is high. 3. The isoelectric points of pig, cow, horse, and rabbit antibodies have been determined. 4. In horse sera prolonged immunization is accompanied by the formation of another antibody component of lower mobility

NEUTRALIZATION OF THE AGENT CAUSING LEUKOSIS AND SARCOMA OF FOWLS BY RABBIT ANTISERA

Neutralizing antibodies against fowl tumor agents can be produced in rabbits by injection of heavy materials obtained from chicken tumor. Similar sediments from normal chicken spleen produce no neutralizing antibodies. The complement-fixing antibodies produced by both materials are unrelated to the neutralizing antibodies

SPECIFIC FRACTIONATION OF HUMAN ANTIDEXTRAN ANTIBODIES : II. ASSAY OF HUMAN ANTIDEXTRAN SERA AND SPECIFICALLY FRACTIONATED PURIFIED ANTIBODIES BY MICROCOMPLEMENT FIXATION AND COMPLEMENT FIXATION INHIBITION TECHNIQUES

Human antidextran of one individual, absorbed specifically on sephadex, was fractionated into two populations of antibody molecules by successive elution with oligosaccharides of the isomaltose series of increasing size. The purified antibody fractions and some whole antidextran sera were found to fix complement with dextrans of molecular weight of 195,000 and above. It could be demonstrated by quantitative microcomplement fixation inhibition assays that the antibody eluted with isomaltotriose had a higher affinity for smaller oligosaccharides relative to isomaltohexaose, indicating a high content of antibody molecules with smaller combining sites, while with the second fraction, eluted with isomaltohexaose, the small haptens were very poor inhibitors and the larger oligosaccharides inhibited readily, presumably due to a higher proportion of molecules with larger combining site size. Assays of similarly prepared fractions, obtained from earlier bleedings of the same individual (1), with inhibition of complement fixation were in good agreement with those obtained by inhibition of precipitation. The two purified antidextran fractions were shown to differ with respect to their complement-fixing capacity. The fraction with molecules with smaller size-combining sites fixed only about half as much complement per unit antibody N as did the fraction containing largely molecules with larger combining sites suggesting that the strength of complement fixation is affected by the strength of the antigen-antibody interaction

CHEMICAL AND IMMUNOLOGICAL STUDIES ON THE AGENT PRODUCING LEUKOSIS AND SARCOMA OF FOWLS

The activity of the agent producing sarcoma or leukosis in material deposited by high speed centrifugation is the same as that of the original crude extracts. Material sedimentable at high speed containing approximately 10–5 mg. N produces tumors at the site of injection. Small quantities of material sedimentable at high speed are present in normal chicken sera, and about twice as much in leukemic sera (strain 1). Normal chicken and mouse spleens and all other human and mouse tissues examined contain large amounts of material sedimentable at high speed. Extraction of leukemic blood cells with saline yields little additional virus. The washed cells readily produce leukosis even after irradiation with amounts of x-rays sufficient to destroy the leukemic cells but not the virus. Freezing at –60°C. preserves the activity of the high speed deposits for at least 6 months. Addition of 5 per cent of saturated Na2SO4 solution slightly delays deterioration of high speed deposits in the ice box. Most of the agent measured by inoculation of chickens and the fraction sedimentable at high speed measured by its nitrogen content is precipitated by one-third saturation with sodium sulfate

IMMUNOCHEMICAL STUDIES ON BLOOD GROUPS : LIV. CLASSIFICATION OF ANTI-I AND ANTI-i SERA INTO GROUPS BASED ON REACTIVITY PATTERNS WITH VARIOUS ANTIGENS RELATED TO THE BLOOD GROUP A, B, H, LEa LEbAND PRECURSOR SUBSTANCES

Quantitative precipitin assays were carried out with eleven anti-I and five anti-i cold agglutinin sera. Their reaction patterns with precursor substances and with certain antigens related to the A, B, H, Lea, and Leb substances revealed at least six types of I and four types of i specificity. All of the I and i determinants appear to be represented in varying amounts on that fraction of the ovarian cyst precursor substance OG which was precipitated by 20% ethanol (OG 20% 2X). Only some of these determinants can be detected in the first periodate oxidation stages of A, B, and H substances, in cow substances, and in hydatid cyst fluid

CHEMICAL STUDIES ON BACTERIAL AGGLUTINATION : III. A REACTION MECHANISM AND A QUANTITATIVE THEORY

1. By the application of an absolute, quantitative microchemical method for the estimation of agglutinins, precise data have been obtained on the course of the agglutination of Type I pneumococcus by homologous anticarbohydrate. 2. Within the limitations imposed by the necessity for the agglutination reaction to take place at the bacterial surface, the reaction is shown to be analogous to the precipitin reaction and subject to the same laws. 3. The entire process of a typical instance of specific bacterial agglutination has been quantitatively accounted for on a purely chemical basis and expressed in the form of equations derived from the law of mass action. 4. Experimental verification of predictions based on the theory has shown a fundamental difference between this instance of specific bacterial agglutination and the commonly adduced analogies, and necessitated a revision of current conceptions regarding the rôle of electrolytes and of physical forces in the reaction

A QUANTITATIVE THEORY OF THE PRECIPITIN REACTION : V. THE REACTION BETWEEN CRYSTALLINE HORSE SERUM ALBUMIN AND ANTIBODY FORMED IN THE RABBIT

1. The reaction between crystalline horse serum albumin and homologous antibody in rabbit sera is quantitatively accounted for by expressions similar to those derived from the law of mass action for other immune precipitating systems. 2. The reaction of an azo dye prepared from crystalline serum albumin by coupling with diazotized R-salt-azo benzidine was also studied with homologous antibody and anti-serum albumin. 3. Quantitative data obtained on cross reactions with the two antigens differ markedly from data on the corresponding reactions in the egg albumin system and indicate that tyrosine and perhaps histidine, while important in determining the serological specificity of egg albumin, have little connection with the specificity of serum albumin. 4. Calculations are made of the equivalent composition of the specific precipitate at various reference points in the reaction range

CHEMICAL STUDIES ON BACTERIAL AGGLUTINATION : I. A METHOD

1. A method, conforming to the criteria of quantitative analytical chemistry, is described for the estimation of the agglutinin content of antisera. Examples are given of the application of the method to various antipneumococcus sera. 2. This new, absolute method is discussed with regard to its relation to the commonly used relative methods

STUDIES ON HUMAN ANTIBODIES : VIII. PROPERTIES AND ASSOCIATION CONSTANTS OF HUMAN ANTIBODIES TO BLOOD GROUP A SUBSTANCE PURIFIED WITH INSOLUBLE SPECIFIC ADSORBENTS AND FRACTIONALLY ELUTED WITH MONO- AND OLIGOSACCHARIDE

Human antibodies to blood group A substance were purified by absorption on columns of insoluble polyleucyl hog blood group A + H substance and eluted first with N-acetylgalactosamine and then with an A active reduced pentasaccharide ARL0.52. The γM and γG antibodies in these eluates were separated by density gradient centrifugation. The antibodies were studied for their relative capacities to be inhibited by various blood group A active oligosaccharides. Antibodies eluted by the N-acetylgalactosamine could be inhibited by N-acetylgalactosamine, as well as by lower concentrations of A active tri- and pentasaccharides, while those eluted by the pentasaccharide ARL0.52 could only be inhibited by the two oligosaccharides, but not by N-acetylgalactosamine, indicating that the N-acetylgalactosamine eluate had more antibodies with smaller size combining sites than the ARL0.52 eluate. Measurements by equilibrium dialysis gave values ranging from 2 x 103 to 1 x 105 M–1 and the values obtained with the ARL0.52 eluate were somewhat higher than those with the GalNAc eluate. Only one of three anti-A sera had γM anti-A in the ARL0.52 eluate, while all three had γM in the N-acetylgalactosamine eluate. Data on the precipitating, hemagglutinating, complement fixing, hemolytic properties of the eluted antibodies, and of their content of κ and λ light chains are given