Justine S. Garvey

Molecular forms of antigen in the circulation following immunization

Abstract Soluble antigen, 35 S-BSA, at 5 hr after intravenous injection into rabbits exists in five different molecular forms in the circulation: (1) intact antigen, (2) modified antigen having a different electrophoretic mobility from the intact antigen, (3) intact antigen or an antigen fragment complexed with IgG antibodies, (4) fragmented antigen with a smaller molecular weight than the intact antigen, (5) fragmented antigen associated with RNA or oligoribonucleotides. By molecular weight difference, the antigen fragments-RNA materials were resolved into three different fractions. Heterogeneity, and varying ionic properties for each fraction were shown by electrophoresis. Base analysis of each fraction revealed a qualitative difference in their RNA components. None of the antigen fragment-RNA materials contained the four common bases of RNA. The cathodic properties of some, and the absence of the four common bases of RNA in all antigen fragment-RNA fractions suggest that the linkage for some of the antigen fragments is to a complex of RNA or oligoribonucleotide and basic protein, rather than as a linkage to the moiety of RNA alone. Further evidence was given for the presence of low molecular weight antigen fragment-nucleoprotein and for a cellular origin other than the peripheral blood, since they have also been found in lymphoid tissues, such as spleen and lymph nodes.

The physicochemical characterization of the ribonucleic acid-antigen complex persisting in the liver of immunized rabbits☆

Abstract Antigen fragments were found associated with S-RNA obtained from the livers of rabbits immunized against S 35 -BSA. Ecteola-cellulose chromatography of S-RNA complex revealed three peaks. Study of the distribution of antigen fragments in the different liver fractions showed a redistribution of the antigen fragments during the immunization. A similar redistribution of the antigen fragments was also observed with S-RNA complex as isolated from RNP fractions and also with n-p ∗ complexes. A steady fall in radioactivity with time was observed with RNP fractions and n-p ∗ complexes, while the radioactivity in S-RNA complexes reached the maximum on the second day after the injection of the radioactive antigen. Column chromatography of n-p ∗ complexes on DEAE-cellulose showed that the n-p ∗ complexes may be resolved into six peaks. A slow transition in the relative amounts of radioactivity from the fast-moving zones to slow-moving ones occurred during the immunization. Peptide mapping of n-p ∗ complexes revealed a definite pattern of antigen fragmentation, with the maximal radioactivity present in histidine and/or tyrosine containing peptides.

The use of a nucleopeptide fraction from injected rabbits to study the nature of antigen fragment-RNA association

Abstract A nucleotide peptide ( n − p ) fraction was obtained from liver tissue of ‘normal’, uninjected rabbits and rabbits injected with either 35 S-sulfanilate-azo-BSA or 3 H-aniline-azo-BSA. The n − p of each was fractionated on Sephadex G-50, and since ‘C’ represented a highly-radioactive, UV-absorbing fraction as obtained from the ‘injected’, it was studied further. In a ‘peptide map’ there were 4, UV-absorbing anodic components containing radioactivity, and it was the presence of the latter in the ‘injected’ that distinguished it from the ‘normal’. 3 H and 35 S as labels, in the injected antigen, of a neutral and a negatively-charged group respectively, were present in the same n − p components following high voltage electrophoresis. The latter is strong evidence for covalent linkage between nucleotide and antigen fragment. In addition to three anodic components, an equal number of components having slight cathodic mobility, were biologically-active, substituting at about a 1000-fold less concentration for antigen in ‘triggering’ an anamnestic response in vitro . The present findings are discussed with reference to the role of antigen in protein (antibody) synthesis.

CHARACTERIZATION OF RNA-ANTIGEN COMPLEXES*

There is still a great lack of information about the in vivo fate of antigen within cells despite the enormous amount of interesting information being contributed in the field of cell immunology. One reason for the gulf seems to be that most ideas persist from, or at least are largely conditioned by, work performed several decades ago with cellular antigens (bacteria and blood cells). Although these agents were the basis for immunology applied to the successful prevention and treatment of disease, a soluble antigen is preferred for investigations of subcellular handling of antigen and its interaction with cellular constituents. Another reason for slow progress in learning about the fate of antigen is the difficulty of distinguishing peripheral events from principal aspects of antigen handling and of concentrating on the role of the latter in the immune response. Separation of functions poses technical difficulties while working in vivo, but the in vitro system has limitations inherent in the lack of total body function. For these implied reasons, we have preferred soluble antigens without adjuvants in studying metabolized antigen and a combined in vivo and in vitro approach. This paper summarizes some of our findings.

On the Role of Antigen Fragments and RNA in the Immune Response of Rabbits to a Soluble Antigen

The purpose of some early studies from this laboratory was to isolate, by mild chemical procedures, antigen material† that had been retained in the tissues of an immunized rabbit. Liver tissue was chosen for the technical reason that the quantity of retained antigen was greater in this tissue than in any other tissue. When liver brei was fractionated with sucrose, antigen material was concentrated in the soluble fraction. Further fractionation to remove non-antigen material was effected by absorption on Dowex-2 resin and subsequent elution with sodium salicylate. Ribonucleic acid was present with the concentrated antigen, and when further fractionation was carried out with 50% ammonium sulfate, the nucleic acid and antigen remained in the soluble fraction with an Sw value of 3.6 to 4.3 (Garvey and Campbell, 1957). The fraction that contained antigen and RNA was at least 200-fold more active for in vitro anaphylactic sensitization, i.e., in the Schultz-Dale reaction than the original antigen and was tentatively regarded as a complex since neither component was dialyzable unless denatured by heat or alkali. Studies carried out over the past dozen years have amplified and extended these initial findings but determining their significance in the immune reaction still requires much work.

Anti-SRBC Plaque-Forming Cells in Liver, Spleen and Hepatocyte Cell Suspensions of Rabbits and Mice

In a series of experiments in rabbits and mice, we have demonstrated that hepatocytes, isolated at a purity exceeding 98% from immunized animals, possess an immunological activity against the immunizing antigen, sheep red blood cells. Hepatocytes formed hemolyzing plaques as assayed by the Jerne technique, and hepatocyte culture fluids showed both hemagglutinating and hemolyzing activity as well as the presence of immunoglobulin components in immunoelectrophoretic patterns.

Comparative Studies of Antigen Injected into Adults and Neonatal Rabbits

Tolerance induced in newborn rahhits (l–4) by an injection of soluble protein antigen resulted in a state of specific unresponsiveness that was dependent in duration upon the amount of antigen injected at birth. Influenced by these findings Medawar (5) modified an earlier definition of tolerance to read: “if an animal is exposed to an antigen before it has developed the capacity to react against it, then the development of that capacity is delayed and, in the continued presence of antigen, can be indefinitely postponed.” Within the past decade, additional investigations have confirmed and extended the earlier findings, but the lack of information on antigen handling by the neonate and the questionable persistence of antigen during maturation provide justification for the present studies.

Prolonged antibody production in rats after single injection of KLH coated on bentonite.

Abstract KLH coated on bentonite stimulated a high titer and a prolonged production of anti-KLH antibody. After one injection of 100 μg or 300 μg KLH coated on bentonite the anti-KLH titer detected one year later by passive hemagglutination was equal to 8200 and 20,480 respectively.