M.H.V. Van Regenmortel

Antigenic relationships between strains of tobacco mosaic virus

Abstract The serological relationships between six strains of tobacco mosaic virus were studied with antisera from 40 rabbits bled at regular intervals during at least 8 mo. The extent of cross reactivity between strains was expressed by a serological differentiation index (SDI) equal to the difference between homologous and heterologous titers denoted as Neg Log 2 . The extent of cross reactivity between two strains was determined in reciprocal serological tests, using antisera against each of the two strains. Average SDI values calculated from a large number of bleedings taken from different animals agreed closely with the corresponding SDI values calculated from reciprocal serological tests. The intraclass correlation coefficient between two sets of SDI values obtained in reciprocal serological tests was r ′ = 0.95. A correlation coefficient of r = 0.75 was calculated between the serological relatedness expressed as SDI values and the extent of sequence homology in the coat protein of different strains.

Isolation of specific antibody under conditions of low ionic strength

Specific rabbit and goat antibodies to tobacco mosaic virus, bovine serum albumin and human gamma globulin have been isolated by acid dissociation from the antigen in the absence of salt. Maximum recovery of 96-100% of active antibody occurred at pH 2.8-2.9. The amount of antibody recovered within any given pH range was highly reproducible. The average antibody affinity was found to be correlated with the pth of dissociation and to decrease exponentially with the length of exposure to acid conditions.

Immunochemical studies of tobacco mosaic virus--II. Univalent and monogamous bivalent binding of IgG antibody.

Abstract Binding experiments with tobacco mosaic virus and IgG antibody were analysed by two types of Scatchard plot. In a large antibody excess the IgG molecules were bound univalently and the antigenic valence of the virus was 780.At lower antibody-antigen ratios, IgG was bound in monogamous bivalent fashion and the same antigenic valence (800) as previously observed with specific Fab fragments was found. The biphasic nature of the curves was considered to be due to a change-over from univalent to bivalent binding. Association constants calculated in the region where IgG binds univalently were considerably lower than for bivalent binding.

Immunochemical studies of tobacco mosaic virus—III.: Demonstration of five antigenic regions in the protein sub-unit

Abstract The antigenic determinants of the protein sub-unit of tobacco mosaic virus have been studied by inhibition of complement fixation experiments. The viral sub-unit has been found to possess five epitopes. Three of them situated in tryptic peptides 1.4 and 8 are cryptotopes, i.e. epitopes absent from the outer surface of the assembled viral capsid. Two other epitopes situated in tryptic peptides 1 and 12 are located in a region that is recognized by both anti-sub-unit and anti-viral antibodies. When the results are analyzed in terms of the three-dimensional structure of the viral sub-unit, it appears that all the antigenic reactive regions occupy highly accessible locations on the surface of the protein.

Immunochemical studies of tobacco mosaic virus--I: refutation of the alleged homogeneous binding of purified antibody fragments.

Abstract Binding experiments with tobacco mosaic virus and specific Fab fragments were analysed by means of two types of Scatchard plot. The first type, which makes no assumption regarding antigen valence, yielded a series of curves which, when extrapolated, revealed that the effective antigenic valence of the virus was 800. The various plots obtained with different Fab concentrations showed that the affinity constants of TMV antibodies depended on antibody concentration, and they did not substantiate earlier claims that TMV antibodies are homogeneous. The second type of Scatchard plot, which is commonly used for monovalent haptens, yielded a series of lines, the slope of which varied with Fab concentration. The linearity of these plots appears to be an artefact produced by the large antigen valence of tobacco mosaic virus, and does not indicate homogeneity of association constants. The forward binding reaction between Fab and TMV is practically irreversible and is largely dependent on an increase in entropy. It appears that previous workers who investigated this system and reported that anti-TMV antibodies are homogeneous were misled because they assumed an incorrect antigenic valence and used equations that are unsuitable for antigens with a very large valence.

Purification by immunoadsorption and immunochemical properties of histone H3

Antibodies to histones represent a powerful tool for investigating the structure of chromatin and chromosomes [ 11. The distribution of histones in chromatin and in nucleosomes has been studied by complement fixation [2,3], immuno-electron microscopy [4], radioimmunoassay [S] and immunofluorescence [6]. It has been found, for instance, that histones Hl and H2B are more exposed in rat liver chromatin than histones H3 and H4 [3], and that more than 90% of the nucleosomes contain histone H2B [4]. The availability of histone antisera raises the possibility of locating the immunologically reactive regions of histones [7,8], and of preparing the corresponding antibody fractions. Such antibodies directed against well-defined portions of histone molecules could be used for determining the ejrtent to which a particular histone is buried inside nucleosomes or is complexed with other chromatin components. In the present report we describe the location of three antigenic determinants in histone H3 from chicken erythrocytes, as well as the purification of this histone by immunoadsorption using antibodies to one of the determinants.

A new method for determination of sedimentation constants of viruses

Abstract New methods are described for the determination of sedimentation constants of viruses in columns layered over gradients of sugar by which unpurified virus preparations can be analyzed, the boundary position being determined by titration or serological measurements. For the latter the gel-precipitin technique proved most suitable. Experimental results are presented for the hemocyanin of Burnupena cincta , the MEF 1 strain of type 2 poliovirus and turnip yellow mosaic virus.

Immunochemical studies of tobacco mosaic virus--IV. Influence of single amino acid exchanges on the antigenic activity of mutant coat proteins and peptides.

Abstract The antigenic properties of tryptic peptides of wild type TMV vulgare have been compared with those of equivalent mutant peptides presenting a single amino acid exchange. Mutants with exchanges at positions 5, 20, 63, 65, 107, 140 and 156 in the sequence of the coat protein possess altered immunochemical properties. Some of the exchanges affect the antigenic reactivity of TMV protein because they are located within an antigenic determinant, whereas others have an influence because they alter the conformation of the polypeptide chain. The results point to the value of combining various approaches for elucidating the antigenic structure of TMV protein.

Three antigenic determinants in histone F3 from chicken erythrocytes

Abstract Antibodies to purified chicken erythrocytes F3 histone were induced by immunizing rabbits with histone-RNA complexes. These antibodies were tested in complement fixation experiments with the whole histone and with three fragments obtained by cyanogen-bromide cleavage. None of the three fragments alone formed complement-binding complexes at a serum dilution (1:300) which gave 90% complement fixation with intact F3 histone. However, when two or more of the fragments were tested in different combinations, up to 60% complement fixation was obtained. All three fragments inhibited to various degrees the complement fixation of F3 histone with its homologous antiserum.

Purification and properties of tomato spotted wilt virus

Abstract Tomato spotted wilt virus was purified by chromatography on columns of calcium phosphate, precipitation with polyethylene glycol, density gradient centrifugation, and ascending zone electrophoresis in a sucrose density gradient. Examination of the virus by electron microscopy revealed particles of different shapes in different suspending media. Particles fixed with glutaraldehyde were spherical and somewhat flattened and had a corrected diameter of 85 nm. Antisera with a titer of 1 128 against the virus were prepared which did not react with plant antigens. The virus had a sedimentation coefficient s20,w of 530 S and, when fixed in 0.5% glutaraldehyde, it had an electrophoretic mobility at pH 7 of −19.8 × 10-5 cm2 V−1 sec−1.