Specificity of antigenic fractions of tuberculin.

Purified protein derivative from Mycobacterium bovis was separated into 6 fractions by electrophoresis at 1500 V/40 mA in pH 4.2 acetic acid-pyridine buffer. Further purification of one of the fractions on Sephadex G 25 column and by acid hydrolysis yielded antigen "PS" eliciting tuberculin reaction only in animals vaccinated with M. bovis BCG but not in those sensitized with M. avium and some fast-growing mycobacteria. The specificity of antigen "PS" was confirmed in vitro: the antigen induced blastic transformation only in lymphocytes of guinea pigs sensitized with M. bovis BCG.     

Two antigenic sites of tissue transglutaminase.

The immunization of rabbits with purified guinea pig liver transglutaminase resulted in the appearance of two antibody populations against the enzyme: one which reacted only with the Ca2+-enzyme complex and another which reacted with the intact as well as the Ca2+-enzyme. The Ca2+-induced confomrational change of the enzyme molecule exposes a new antigenic determinant which initiates the production of a specific antibody population. When the glutamine substrate of the enzyme was a dipeptide, the result of the interaction of the Ca2+-enzyme and its isolated specific antibody was an apparent activation of the catalytic activity. However, when protein substrates were used, an inhibition was observed. The characterization of the mechanism of the activation and the inhibition has led to the conclusion that the consequence of the interaction of Ca 2+-enzyme and its specific antibody is not only a limited steric hindrance of the active center but, besides that, a stabilization of the otherwise labile Ca2+-enzyme. The other antibody population reacts with both forms of the enzyme and its inhibitory effect, which has been observed in each assay, could be due to a prevention of the Ca2+-induced formation of the active enzyme.     

The antigenic structure of bovine serum albumin. Evidence for multiple, different, domain-specific antigenic determinants.

Using antiserum to native bovine albumin and antigenically active fragments of the protein, we have isolated antibodies directed to each of the three domains and to several subdomains of the albumin molecule. Using albumin and these fragments as inhibitors of the reaction between 125I-albumin and any given antibody population, we have demonstrated that: (a) each domain of albumin is antigenically distinct from each of the other domains; (b) each domain possesses a minimum of two different antigenic determinants; and (c) the entire albumin molecule possesses a minimum of six different, nonrepeating, antigenic determinants.     

Predicting antigenic sites on proteins.

The ability to predict antigenic sites on proteins is of major importance for the production of synthetic peptide vaccines and synthetic peptide probes of antibody structure. Many predictive methods, based on various assumptions about the nature of the antigenic response have been proposed and tested. This review will discuss the principles underlying the various approaches to predicting antigenic sites and will attempt to answer the question of how well they work.     

Existence of common antigenic sites in tropomyosins.

1. Tropomyosins were extracted from vertebrate and invertebrate muscles, and their immunolo;ical characteristics were compared using antisera against tropomyosins from chicken skeletal and cardiac muscles. 2. Antigenic sites common to those of chicken skeletal muscle tropomyosin were found in all the tropomyosins tested, although the reactions of these common antigenic sites in an immunodiffusion test were weak in tropomyosins from phylogenetically distant animals. 3. An immunological difference was found between alpha-tropomyosins from chicken cardiac muscle and rabbit cardiac muscle. Thus they had specific antigenic sites in addition to the common ones. 4. A component was found in a 1 M KCL extract of Tetrahymena pyriformis which reacted with antiserum against chicken skeletal muscle tropomyosin.     

Exposure of histone antigenic determinants in chromatin.

The exposure of antigenic determinants of histones present in "native" chromatin was studied by: (1) testing their ability to elicit anti-histone antibodies and (2) measuring their ability to interact with anti-histone sera. To this end, antisera specific to purified histone fractions and to purified rat liver chromatin were elicited in rabbits. The anti-chromatin sera did not react with pure histone fractions and pure histone fractions F2b, F3, F2a1, and F2a2 failed to inhibit the complement fixation resulting from the binding of anti-chromatin to chromatin. These results suggest that in native chromatin, determinants in these histones are not immunogenic. Histone F1, however, inhibited the reaction between chromatin and anti-chromatin. Antisera elicited by histone fractions reacted weakly with "native" chromatin. The maximal complement fixations (obtained with 5-10 mug of chromatin DNA) were as follows: 60% with anti-F2b, 20% with anti-F1 and anti-F3, and less than 5% with either anti-F2a1 or anti-F2a2. Studies of the interaction between anti-histone antibodies and chromatin in which chromatin was used as an immunoadsorbent indicated that antibodies against different histones were adsorbed to a different degree by the same amount of chromatin. Differences in the immunoadsorbing capacity between sonicated and nonsonicated chromatin were found. Quantitative adsorbtion studies revealed that in the "native" chromatin structure, antigenic determinants of F1 and F2b were more available to interact with homologous antibody than those of F3 and F2a1 and that determinants in F2a2 were the least available. It could be calculated that the "equivalent antigenicity" of the histones in chromatin was 9.6% for F1, 3.2% for F2b, and 0.90% for F3 and F2a1. Upon sonication these values did not change for F1 but increased two-, three-, and fourfold for F2b, F3, and F2a1, respectively. Digestion of chromatin with trypsin totally abolished the ability of chromatin to adsorb anti-histone antibodies.     

Norovirus GII.4 strain antigenic variation

Noroviruses are the principal cause of epidemic gastroenteritis worldwide. Multiple reports have concluded that the major capsid proteins of GII.4 strains, which cause 80% of norovirus infections worldwide, are evolving rapidly, resulting in new epidemic strains. Surrogate neutralization assays using sera from outbreaks and from immunized mice suggest that, as with influenza virus, antigenic variation maintains GII.4 persistence in the face of human population herd immunity. To test this hypothesis, mice were hyperimmunized with virus-like particles (VLPs) representing an early (GII.4-1987) and a contemporary (GII.4-2006) GII.4 strain. Anti-GII.4-1987 IgG monoclonal antibodies (MAbs) strongly reacted with GII.4 VLPs derived between only 1987 and 2002. Ligand binding blockade was more efficient with GII.4-1987 and GII.4-1997 VLPs than with GII.4-2002. Anti-GII.4-2006 IgG MAbs recognized either a broad panel of GII.4 VLPs (1987 to 2006) or a subset of contemporary (2004 to 2006) VLPs. Most 2006 antibodies did not recognize or only poorly recognized GII.4 VLPs of 2007 or 2008, documenting rapid antigenic evolution of GII.4 capsids. Generally, 2006 MAbs blocked homotypic VLP-ligand binding but were unable to block VLPs representing strains primarily circulating during or earlier than 2002. These analyses demonstrate that both subtle and significant evolutionary change has occurred within antibody epitopes between epidemic strains, providing direct evidence that the GII.4 noroviruses are undergoing antigenic variation, likely in response to herd immunity. As with influenza virus, HIV, and hepatitis C virus, norovirus antigenic variation will significantly influence the design of efficacious vaccines and immunotherapeutics against these important human pathogens.     

Human glomerular basement membrane. Heterogeneity of antigenic determinants.

Human glomerular basement membrane was solubilized by digestion with proteolytic enzymes and immunoreactive components were quantitated and characterized by using rabbit antibodies raised against the particulate membrane. A number of antigens were demonstrated but they did not separate on gel filtration. However, two antigenic components in a collagenase digest of the membrane could be separated and isolated by Sepharose 6B chromatography. Chemical characterization suggests that both fragments are noncollagenous glycopeptides (molecular weights approx. 1,000,000 and 60,000--200,000, respectively).     

Isofocusing of antigenic small nuclear ribonucleoproteins. I. Compositional analysis

This report describes the behavior of antigenic small nuclear ribonucleoproteins (snRNPs) in native isofocusing gels. These RNA-protein complexes exhibited true isofocusing characteristics only when in the complexed form. Deproteinized snRNAs migrated to pH ranges which varied according to the pH of the application site. Immunological assays using lupus sera which recognized the La, Sm, and RNP determinants on these snRNPs established that the La and the Sm/RNP antigens segregated to pH 4.7-4.9 and 5.5-7.5, respectively. RNase digestion of these snRNPs did not alter the isofocusing migration of either the Sm or the La determinants. These antigenically active fractions contained the appropriate protein and RNA species shown by immunoprecipitation studies to associate with these antigenic determinants. The isofocusing fractions containing the uridylic acid-snRNPs were fully immunoprecipitable by anti-Sm sera, confirming their particulate integrity after isofocusing.     

Isofocusing of antigenic small nuclear ribonucleoproteins. II. Preparative isolation

In a companion report (T.B. Okarma, W.S. Schrier, and R. Feinbaum, 1985, Anal. Biochem. 147, 27-37) the behavior of small nuclear ribonucleoproteins (snRNPs) in native isofocusing gels was characterized. This communication extends those findings and describes a gentle procedure for the preparative isolation of snRNPs in native form from cultured murine L-5178y leukemia cells using sucrose density gradient centrifugation, preparative isofocusing, and gel filtration chromatography. Isofocusing in granulated gels separated intact uridylic acid (U)-snRNPs from tRNA and La RNPs. The U-snRNPs remained immunoprecipitable by lupus antisera throughout fractionation. The final product obtained in 2% yield contained primarily U1 and U2 snRNAs and lesser amounts of U3, U4, U5, and U6, along with the core U-snRNP polypeptides A-G. The core polypeptides displayed apparent pI's which ranged from 4.5 to 9.5 when analyzed by two-dimensional gel electrophoresis. Proteins B (28,000), D (16,000), and E (13,000) exhibited isoelectric variants. The Sm determinant proteins B' (28,000) and E (13,000) isofocused as basic peptides with apparent pI's of 9.5 and 8.5, respectively. The purity of the final fractions compared well with that of immunoprecipitates and the procedure reproducibly generated yields of native snRNPs sufficient for in vitro studies of their biological function.     

Genetic basis of Neisseria gonorrhoeae lipooligosaccharide antigenic variation.

Neisseria gonorrhoeae lipooligosaccharide (LOS) undergoes antigenic variation at a high rate, and this variation can be monitored by changes in a strain's ability to bind LOS-specific monoclonal antibodies. We report here the cloning and identification of a gene, lsi-2, that can mediate this variation. The DNA sequence of lsi-2 has been determined for N. gonorrhoeae 1291, a strain that expresses a high-molecular-mass LOS, and a derivative of this strain, RS132L, that produces a truncated LOS. In the parental strain, lsi-2 contains a string of 12 guanines in the middle of its coding sequence. In cells that had antigenically varied to produce a truncated LOS, the number of guanines in lsi-2 was altered. Site-specific deletions were constructed to verify that expression of a 3.6-kDa LOS is due to alterations in lsi-2.