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The term ‘antigen’ refers to any entity, whether a cell, a macromolecular assembly or a single molecule, which can elicit an immune response in a competent, vertebrate host and be recognized specifically by the products of that immune response. The ability of antigens to react specifically with complementary antibodies is known as ‘antigenic reactivity’ or ‘antigenicity’, while their capacity to generate an immune response is called ‘immunogenicity’. It should be noted that immunogenicity is not an intrinsic property of the antigen but a relational property that depends on the gene repertoire and regulatory mechanisms of the host being immunized and which has no meaning outside the context of the host (Berzofsky 1985). For instance, mouse serum albumin is an antigenic protein that is immunogenic in the rabbit but not normally in the mouse because of the regulatory mechanism known as immunological tolerance.

Isolates of Rice yellow mottle virus (RYMV) were typed at the molecular level through the sequences of the open reading frame (ORF) 4 (coding for the coat protein) and ORF1 (coding for the movement protein), and serologically by means of polyclonal and monoclonal antibodies. The overall patterns of diversity shown by molecular and serological analyses were similar: East-African isolates differed from West-African ones, and the West-African isolates from forest differed from the savannah ones. Each major strain had a different serological profile. However, molecular typing was more discriminating than immunological typing since several sequence variants belonged to the same serotype. In rare instances, there were explainable discrepancies between molecular and serological typing. Two amino acids at positions 115 (alanine vs threonine) and 191 (valine vs threonine) consistently discriminated between the major serotypes. These positions were located in antigenic sites as revealed by Spot-scan method and were recognised by discriminating monoclonal antibodies. One shared epitope, lying within a conserved region, may be responsible for the cross-reactivity between RYMV isolates. A rationale for the correlation between molecular and immunological typing of RYMV and other sobemoviruses is proposed.

It is widely believed that all biological phenomena can be reduced to chemistry and physics. Such a reductionist view disregards the fact that complex biological systems have relational (also called emergent) properties that their constituents lack and that cannot be deduced or predicted from the properties of the isolated components. When the individual components of the immune system are studied in isolation, many interconnections are lost and it is not possible to understand how the system functions at the level of the organism as a whole. Our increasing knowledge of the antigenic structure of viral proteins has also been of little help for improving the immunogenicity of individual viral epitopes and for enhancing their capacity to elicit a protective immune response against viral infection. When molecular design principles are used to optimize the binding properties of a synthetic peptide epitope with respect to one neutralizing monoclonal antibody, this does not ensure that the peptide, when used as immunogen, will be able to induce neutralizing antibodies that protect against disease. A reductionist approach does not provide the information required for designing peptide immunogens that will elicit neutralizing rather than non-neutralizing antibody responses.

Whereas the concentration of a biomolecule simply refers to the amount of chemical substance per unit of volume, its active concentration refers to a relational parameter that has meaning only with respect to the molecule's ability to interact specifically with one particular ligand. When proteins are studied in a biological context, it is the biologically active concentration that is relevant, and not the total concentration of correctly and incorrectly folded molecules. Using a biosensor instrument the concentration of active biomolecules in a preparation can be measured by injecting the preparation at different flow rates onto a sensor chip surface presenting a high concentration of a specific ligand. The method can be used under conditions of partial mass transport limitation and does not require a pre-established standard curve. When the method was used to measure the active concentration of several recombinant proteins it was found that the active concentration was much lower than the nominal concentration determined by conventional methods. The active concentration also depended on the ligand used in the binding assay, reflecting the fact that active concentration can only be defined with respect to one specific probe. Such discrepancies in concentration values, if undetected, may lead to erroneous conclusions regarding the properties and behaviour of recombinant proteins tested in different assays. Copyright © 1999 John Wiley & Sons, Ltd.

In view of our increased understanding of the molecular basis of immunological recognition, it is commonly believed that it should be possible to apply molecular design strategies to the development of peptide-based vaccines. The stated aim is to transform the development of a vaccine from a trial and error empirical operation into a so-called rational, structure-based process. In the present review, it is argued that it is misleading to oppose rational and empirical approaches in vaccine research since both are needed in the practice of experimental science. Many reasons are given for the view that the molecular design of synthetic vaccines is not a realistic scientific enterprise. The capacity of a peptide to induce a protective immune response depends on many extrinsic factors and regulatory mechanisms of the recipient host which are not amenable to molecular design of the peptide immunogen. It seems safe to predict that the development of peptide-based vaccines will continue to be driven by empirical discovery rather than by so-called rational design.

Kinetics of 3-hydroxyphthaloyl-beta-lactoglobulin-CD4 interaction were evaluated using a biosensor instrument based on surface plasmon resonance. A very fast association (k(a)=2.4+/-0.3x10(6)M(-1)s(-1)) and slow dissociation (K(d)=2.3+/-0.14x10(-4)s(-1)) rate constants were observed indicating the high affinity of the complex. This result together with earlier data, suggest that "structure-specific" requirements must be met to endow acid anhydride modified lactoglobulin with the capacity for high affinity binding to CD4.

Nine pigs were given a single inoculum of 100 microg of the all-D retro peptide corresponding to the immunodominant GH loop encompassing residues 141-159 of capsid protein VP1 of foot-and-mouth disease virus serotype A, sub-type 12. The peptide was conjugated to activated keyhole limpet haemocyanin and oil-adjuvanted before inoculation. The animals were challenged eleven weeks post-vaccination by exposing them to a pig which had been infected with the virus by inoculation. Two naive animals were included in the challenge study as controls. One of the vaccinated animals was completely unprotected and two developed very small lesions. None of the six remaining animals exhibited any clinical signs but two developed antibodies against nonstructural proteins indicating that replication of the virus had occurred. No evidence of replication could be detected in the remaining four animals, either by rise in neutralizing antibody titre or by production of antibodies against non-structural proteins specific for virus replication.

The term specificity is derived from the word species and shares with it an inherent fuzziness based on the absence of sharp boundaries between closely related entities. Antibody specificity is a ternary relational property which refers to the antibody's capacity to discriminate between two or more epitopes. There are no sharp boundaries between the individual overlapping epitopes that constitute an antigenic site and there is also no clear-cut minimum difference in binding affinity or in atomic positions at the epitope-paratope interface that can serve as a yardstick for deciding that two epitopes or two paratopes are the same or not. Immunology shares with the whole of empirical science the need to handle fuzzy sets and concepts and this poses no threat to the unabated further development of immunochemical analysis.

The immunosuppressive cyclic undecapeptide cyclosporin A (CS) exists in various conformers in water. Up to 1 h is needed to reach maximum complex formation after mixing the drug with its receptor, cyclophilin or with a monoclonal antibody. Differences in the ability of CS and its analogs to bind to antibody or cyclophilin have been measured using the BIAcore. These experiments suggest that the rate-limiting step of complex formation is determined by the interconversion between different CS conformers existing in solution. The contribution to antibody binding of individual atomic groups of CS was evaluated by measuring the equilibrium affinity constants of analogs with the BIAcore. When the binding data were analyzed in terms of the known crystallographic structure of the CS/Fab complex, it could be shown that modifications of CS residues located in the central part of the binding site drastically affect affinity, while modifications of residues located at the periphery are more easily accommodated.

Three analogues of the model peptide of sequence IRGERA corresponding to the COOH-terminal residues 130-135 of histone H3 were synthesized, and their antigenicity, immunogenicity, and resistance to trypsin were compared to those of the natural L-peptide. The three analogues correspond to the D-enantiomer, containing only D-residues, and two retro-peptides containing NH-CO bonds instead of natural peptide bonds. The chirality of each residue was maintained in the retro-peptide and inverted in the retro-inverso-peptide. Antibodies to the four peptide analogues were produced by injecting BALB/c mice with peptides covalently coupled to small unilamellar liposomes containing monophosphoryl lipid A. Each of the four peptide analogues induced IgG antibodies of various subclasses. The IgG3 antibodies reacted similarly with the four analogues, whereas antibodies of the IgG1, IgG2a, and IgG2b isotypes showed strong conformational preferences for certain peptides. The retro-inverso-peptide IRGERA mimicked the structure and antigenic activity of the natural L-peptide but not of the D- and retro-peptides, whereas the retro-peptide IRGERA mimicked the D-peptide but not the L- and retro-inverso-peptides. The equilibrium affinity constants (Ka) of three monoclonal antibodies generated against the L- and D-peptides with respect to the four peptide analogues were measured in a biosensor system. Large differences in Ka values were observed when each monoclonal antibody was tested with respect to the four peptides. The use of retro-inverso-peptides to replace natural L-peptides is likely to find many applications in immunodiagnosis and as potential synthetic vaccines.

The different types of epitopes found in viral antigens are described. The BIAcore has been used successfully to map the location of epitopes in viral antigens and several examples of such studies are presented. Knowledge of the affinity of viral antibodies is important for understanding the molecular basis of viral antigenicity and the mechanism of infectivity neutralization by antibodies. The use of the BIAcore for measuring binding constants of viral antibodies is described. Affinity constants of antibodies binding to virus particles are easily obtained using sensor chips on which the virus is immobilized by a first layer of antibodies. Affinity constants obtained with the BIAcore agree closely with values obtained by several other methods of affinity determination. By comparing the binding properties of neutralizing and nonneutralizing viral antibodies with the BIAcore, it should be possible to establish whether there is a relationship between affinity and neutralizing capacity of antibodies. By immobilizing synthetic peptides corresponding to viral epitopes on sensor chips, the BIAcore can also be used to quantitate antibody to specific epitopes present in polyclonal antiserum.

Synthetic peptides conjugated to a protein carrier were used to immunise rabbits in an attempt to produce antisera specific for the different classes of gliadins and for the low M r glutenin subunits of wheat. Eight peptides were selected from regions of low homology situated in the N and C-terminal domains of these proteins. The anti-peptide antisera were assayed gliadin and glutenin fractions of the bread wheat Hardy by immunoblotting after SDS-PAGE separation and by ELISA. Six of the peptides, i.e. N-terminal peptides of α,β-type, γ-type and ω-type gliadins and low M r glutenin subunits and a C-terminal peptide of α,β-type gliadins, induced antibodies that reacted specifically with the cognate proteins. C-terminal peptides of γ-type gliadins induced antibodies that cross-reacted with ω-type gliadins.

Association (ka) and dissociation (kd) rate constants of three monoclonal antibodies raised against tobacco mosaic virus were determined using a biosensor technique based on surface plasmon resonance (BIAcore, Pharmacia). Dissociation rates were constant over the 4-400 nM antibody concentration range whereas apparent association rates decreased over this range probably due to an increased saturation level of the antigen. Affinity constants K calculated from the ratio of ka/kd were in reasonable agreement with values obtained under equilibrium conditions by two standard methods based on enzyme immunoassay.

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In the present study, we developed new turn scales based on the occurrence of amino acids at each of the four positions of a turn using a structural database comprised of 87 proteins. We found that the scales correctly predicted a fraction of the turn regions in proteins with approximately 80% confidence. We used the turn scales for predicting the location of antigenic sites in proteins. The method was developed with the specific aim of predicting only a few peaks for each protein (two or three). We found that it leads to a high level of accurate prediction (70% of correct prediction of known epitopes). Our method should be useful for selecting protein regions to be synthesized in order to produce anti-peptide antibodies cross-reacting with the parent protein.

The antigenic structure of the comovirus bean pod mottle virus (BPMV) was studied using synthetic peptides selected on the basis of the exposed location of certain regions of the viral protein. Three regions of domain A, four regions of domain B and two regions of domain C of BPMV coat protein were studied. Each of four regions were synthesized in the form of linear and cyclized peptides while the others were synthesized as linear peptides only. The peptides were tested for their ability to be recognized by antibodies directed against BPMV. The peptides were also used for producing rabbit antisera, which were tested for their ability to react with various BPMV antigens as well as with the linear and cyclized peptides. All the peptides were found to correspond to epitopes of BPMV coat protein. Several of the antigenic sites of BPMV located on exposed loops of the coat protein occupy positions which correspond to known epitopes in the structurally related picornaviruses. Only in some cases did cyclization sufficiently improve the level of conformational mimicry between peptides and the viral protein to allow cross-reactions between them to be observed.

Summary Eight peptides corresponding to conserved regions of the coat protein of potyviruses were synthesized. All the peptides were recognized by anti-virus or anti-core-virus. Antisera raised to the synthetic peptides were tested with purified viruses and viral antigens present in plant sap. In many cases, the extent of cross-reactivity between different potyviruses was not correlated with the degree of sequence homology between the peptide used for immunization and the corresponding region in the coat protein of the potyvirus tested. An antiserum raised to a peptide of 18 residues containing a highly conserved region was found to react with all seven potyviruses tested.

Sixteen overlapping fragments of the dengue-2 virus envelope (E) protein, expressed as trpE-E fusion products in Escherichia coli, were used to map the epitopes defined by a panel of 20 monoclonal antibodies (MAbs) by immunoblotting. Using this technique, the amino acid sequence of six antigenic domains on the E protein was characterized. Nonneutralizing MAbs were found to define either linear-specific, subcomplex-specific (amino acids 22–58), and complex-specific (amino acids 304–332) epitopes or a subcomplex conformational-dependent epitope requiring the presence of two closely linked amino acid sequences from the E protein, 60–97 and 298–397. Neutralizing MAbs, however, defined either group-reactive epitopes present on two overlapping domains (amino acids 60–135; amino acids 60–205) or type-, subcomplex-, complex-, subgroup-, and group-specific determinants (amino acids 298–397). These neutralizing epitopes were all found to be dependent upon disulfide bridges. Our results suggest that the maintenance of a topographical arrangement of discontinuous antigenic domains in the flavivirus E-protein is necessary to induce neutralizing and protective antibodies.

The association constant of monoclonal antibodies (Mabs) to tobacco mosaic virus has been determined in solution and solid-phase binding assays. The ELISA equilibrium titration method developed by Friguet et al. (1985) was found to be suitable for large antigens such as viruses. In the case of intact IgG antibody, it gave equilibrium constant (K) values ca 30% lower than those obtained by classical solution-phase assay while in the case of Fab', the same values were obtained in both assays. Solid-phase binding assays gave higher K values than solution-phase assays by a factor which varied with the Mab tested (1.5- to 5.4-fold higher). Furthermore, in solution-phase assay, K values were found to depend on the antibody concentration used in the assay. These results confirm the operational nature of antibody affinity constants and indicate that in order to compare the affinity of different Mabs in a meaningful way, it is necessary to use a single technique under standardized conditions.

Four linear synthetic peptides corresponding to residues 12-29, 50-67, 121-138 and 131-147 of the HA 1 subunit of H3 subtype influenza virus (NT/60/68) were tested for their capacity to elicit in vivo peptide-specific CD4+ T cells cross reacting with whole virus. By studying the in vitro peptide proliferative response of lymph node cells from mice sensitized in vivo with free peptides emulsified in complete or incomplete Freund adjuvant, it was found that region 12-29 could be recognized by CD4+ T lymphocytes in the context of H-2k and H-2b, region 50-67 in association with H-2b and region 121-138 in the context of H-2d MHC molecules. Outbred OF 1 mice could recognize regions 50-67 and 121-138. Peptides 50-67 and 121-138 are of potential interest for synthetic vaccine design since they induced in BALB/c (peptide 121-138) and OF 1 (both peptides) mice a CD4+ T cell population that cross reacted with whole virus. The region 50-67 is of particular interest since only few substitutions have been found in this area in natural variants of the H3 virus subtype.

SUMMARYThe antibody response of rabbits immunized with a total histone mixture containing randomly coiled H1/H5, H2A, H2B, H3 and H4 devoid of DNA was investigated in direct and competitive ELISA. The antisera were tested with isolated histones and chromatin and with a series of overlapping synthetic peptides covering the entire sequences of the four core histones and two peptides of H I. It was found that the New Zealand (NZ) white rabbits immunized with the total histone (TH) mixture complexed with RNA produced IgG antibodies reacting with histones and with a number of histone peptides but not with chromatin. The antisera also contained IgG antibodies which bound components that correspond lo common target antigens in autoimmune diseases such as native dsDNA, peptides of Sm-D antigen, ubiquilin, branched peptides of ubiquitinated H2A and poly(ADP-ribose). By competition experiments, it was shown that these antibodies corresponded to non-crossreacting antibody populations. New Zealand rabbits immunized with TH in the absence of RNA or random outbred rabbits immunized with the RNA-complexed histone fraction produced antibodies reacting with histone, chromatin and very few histone peptides. while no activity with non-related antigens was observed. The pattern of reactivity of antisera raised in NZ rabbits with RNA-complexed TH was found lo be very similar to that observed in sera of patients with systemic lupus erythematosus while, in contrast, the antibody response was very different in NZ or outbred rabbits immunized with various native nuclear particles and with individual histones. Altered nucleosome particles rather than native nucleosomes may represent the antigenic stimulus giving rise to autoantibodies.

A number of continuous epitopes of tobacco mosaic virus protein (TMVP) have been defined by the pepscan technique using polyclonal and monoclonal antibodies to TMVP as well as antisera raised against synthetic peptides. In general, the location of continuous epitopes agreed with the results of earlier studies with peptides synthesized by classical methods although there were some notable exceptions. Results obtained with the different types of antibodies used in this study indicated that a homology of three residues was sufficient to give rise to antigenic cross-reactions. In the case of antibodies raised against a peptide conjugated to ovalbumin, some unexpected cross-reactivities could be explained by assuming that antibodies to the carrier molecule recognized homologous tripeptide sequences in TMVP and ovalbumin.

The use of antibodies in immunoaffinity separations represents one of the most specific methods for purifying substances of biological interest. Since the binding affinity of antibody greatly influences its behavior in such separations, it is often important to know the value of the antibody affinity expressed as an equilibrium constant K. The present review discusses the equations used in the quantitative analysis of antigen/antibody interactions and describes currently used experimental methods for measuring K values. Advantages and shortcomings of the solution phase and solid phase approaches used for measuring antibody affinity are discussed.

Genome and Antigenic Variability of Retroviruses.- Genetic Variation in Retroviruses.- Human Immunodeficiency Virus Variation and Epidemiology of Acquired Immunodeficiency Syndrome and Human Immunodeficiency Virus Infection.- Acquired Immunodeficiency Syndrome.- Escape of Lentiviruses from Immune Surveillance.- Visna Virus Genome.- In Vivo and In Vitro Selection of Equine Infectious Anemia Virus Variants.- Genome and Antigenic Variability of Myxoviruses and Paramyxoviruses.- Evolutionary Lineages and Molecular Epidemiology of Influenza A, B, and C Viruses.- Antigenic and Genetic Variation of Influenza A(H1N1) Viruses.- Antigenic Variation among Human Parainfluenza Type 3 Viruses.- Genes Involved in the Restriction of Replication of Avian Influenza A Viruses in Primates.- Newcastle Disease Virus Variations.- Variability of Picornaviruses and Rotaviruses.- Molecular Epidemiology of Wild Poliovirus Transmission.- Virus Variation and the Epidemiology and Control of Rhinoviruses.- Genetic Variability and Antigenic Diversity of Foot-and-Mouth Disease Virus.- Analysis of Rotavirus Proteins by Gene Cloning, Mutagenesis, and Expression.- Virus Hemorrhagic Fevers.- The Molecular Epidemiology of Dengue Viruses.- Hantavirus Variation and Disease Distribution.- Nairoviruses.

Although affinity and kinetic measurements on their own provide useful information regarding the suitability of antibodies for various immunoassays, a thermodynamic analysis provices additional information that throws light on the molecular forces at work in the antigen-antibody interaction. It may then be possible to adjust assay conditions in order to favour either the association or dissociation of antigen-antibody complexes. A tenfold increase in binding affinity (K) corresponds to a free energy change of only 1.4 kcal/mol (5.8 kJ/mol) at 25 °C. This means that K values of 105 M−1 and 1010 M−1 correspond to a free energy change (∆G) of 7.0 and 14.0 kcal/mol respectively. The entire range of affinity constants normally encountered in antigen-antibody interactions, therefore differs by no more than about 7 kcal/mol of free energy change, which is equivalent to only a few hydrogen bonds. In comparison, a single electrostatic interaction corresponds to about 4 kcal/mol of free energy change. A full description of the binding interaction requires an understanding of the change in hydration states of the reactants when the complex forms, and an assessment of the entropic andenthalpic effects of these changes. Contrary to earlier assumptions, it is now clear that antigen-antibody interactions are often accompanied by a large favourable enthalpy which more than compensates the unfavourable entropy.

The specificity of 18 monoclonal antibodies directed to tobacco mosaic virus (TMV) was studied by measuring their ability to bind to viral mutants, to other tobamoviruses, to dissociated viral subunits and to peptide fragments of the viral coat protein. The apparent binding specificity of the antibodies was dependent on the type of enzyme-linked immunosorbent assay used, probably because the antigens were disrupted or denatured when attached to the plastic surface of microtiter wells. The capacity of different monoclonal antibodies to detect single substitutions in the viral coat protein was used to delineate some of the topographic epitopes of TMV. By means of computer-generated images of the surface residues of the viral subunit, it was possible to identify certain clusters of residues involved in binding to some of the monoclonal antibodies. The results clearly illustrate the operational limitations encountered when monoclonal antibodies are used for elucidating the antigenic structure of proteins.

Native, reassociated, and reconstituted core particles from chicken erythrocytes were compared by both biophysical and immunochemical methods. No significant difference between the three types of core particles could be demonstrated by electron microscopy, circular dichroism, or immunochemical analysis with antisera to histone H2B, H2A, and H3. Core particles were also reconstituted with calf thymus non-acetylated H3, H2A, and H2B with either mono-, di-, or tri-acetylated H4 isolated from cuttle -fish testes. The hyperacetylation of H4 did not significantly alter the biophysical characteristics of core particles but it induced several changes in their immunochemical reactivity. Binding to core particles of antibodies specific for H2A, H3, and for the IRGERA (synthetic C-terminal) peptide of H3 was considerably decreased when di- or tri-acetylated H4 was used for reconstitution, whereas binding of H2B antibodies remained the same. Our results suggest that the presence of hyperacetylated H4 within core particles leads to conformational changes that alter the antigenic determinants of several of the histones present at the surface of chromatin subunits. Since histone acetylation is correlated with the open structure of active chromatin, it may become possible to monitor the activity of chromatin by immunochemical methods.

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.

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.

Summary Plant virologists who maintain that the concept of species cannot be applied to viruses argue their case in terms of an obsolete concept of biological species defined by gene pools and reproductive isolation and applicable only to sexually reproducing organisms. In fact, various species concepts have been used by biologists and some of them are applicable to asexual organisms. The rationale for applying the species concept in virology is that viruses are biological entities and not chemicals: they possess genes, replicate, specialize, evolve and occupy specific ecological niches. The following definition is proposed: a virus species is a polythetic class of viruses constituting a replicating lineage and occupying a particular ecological niche. Such a definition of the species category does not and cannot provide a list of diagnostic properties for recognizing members of a particular virus species. It should also be stressed that a single property such as an arbitrary level of genome homology or the extent of serological relationship always fails to establish membership in a polythetic class. A binomial system of nomenclature is advocated in which the vernacular English name of the plant virus is adopted as the species name and the group name is assimilated to the level of genus. Adoption of this system would ensure that a universal classification system based on the classical categories of species, genus, and family becomes possible for all viruses.

Two peptides of eight (T2) and 10 (T1) residues corresponding to the branched moiety of ubiquitinated histone H2A have been synthesized and used for raising specific antibodies in rabbits. Antisera to peptide T1 reacted in ELISA with T1 and with H2A but not with ubiquitin; antisera to peptide T2 reacted with T2 but not with H2A or ubiquitin. When tested in immunoblotting, both peptide antisera reacted with ubiquitinated H2A but not with unconjugated H2A or with ubiquitin. Sera from patients with systemic lupus erythematosus (SLE) were shown previously to react with ubiquitin in ELISA and immunoblotting. When tested for their ability to react in ELISA with synthetic peptides T1 and T2, 96% of the SLE sera (diluted 1:500) that recognized ubiquitin also reacted with peptide T2. Of the SLE sera that did not react with ubiquitin, only 13% possessed antibodies able to bind peptide T2. Antibodies from seven SLE sera, purified on a T2-immunoadsorbent column, were also able to react either with H2A, and in three cases also with ubiquitin.

Conditions suitable for the serological trapping of different plant viruses on electron microscope grids coated with the respective antisera, have been defined. The viruses studied are turnip yellow mosaic, tomato bushy stunt and cauliflower mosaic viruses, and five strains of tobacco mosaic virus. Since the extent of serological cross-reactivity between the TMV strains was known, it was possible to determine how closely related serologically two strains of a virus have to be in order to be trapped on grids coated with the heterologous antiserum. The findings show that optimal conditions for the specific trapping of virions must be determined empirically for each particular virus.

Many studies that are intended to unravel the nature of protein antigenicity actually focus on the phenomenon of cross-reactive antigenicity between proteins and short peptides. As a result, our present knowledge concerns mainly ‘adulterated’, incomplete antigenic sites that have retained only part of their identity after fragmentation of the protein. Nevertheless, this onesided view of protein antigenicity proves to be remarkably useful as many of the practical applications of immunochemical research are based on the exploitation of antigenic cross-reactions.

The application of an enzyme-linked immunosorbent assay to the study of histone antigens is described. This method is much more sensitive than microcomplement fixation tests and allows the interaction between histone antibodies and nucleosomes to be measured directly on the solid phase under a variety of ionic conditions. The results indicate that the enzyme-linked immunosorbent assay is a sensitive assay for investigating different conformational states of chromatin and for detecting low levels of histone antibodies in antisera.

The reactivity of monoclonal antibodies (McAbs) to the coat protein of tobacco mosaic virus (TMVP) with the isolated coat protein, disks, virions and a number of antigenic variants of TMV was tested in eight different ELISA procedures. Although certain McAbs, when used as detecting antibody in the liquid phase, did not react with some of these antigens, they were able to bind to them when used as the capturing antibody on the solid phase. This finding was attributed to the ability of the trapping McAb to induce a complementary conformation in the antigen presented in the liquid phase. In many cases, the reactivity of the McAbs was found to depend on the format of the ELISA. This finding together with the presence of oligomers in viral coat protein preparations made it impossible to map TMVP epitopes on the surface of the viral subunit by means of competitive ELISA.

The degree of antigenic relatedness between two plant viruses is commonly expressed by a serological differentiation index (SDI) which corresponds to the average number of two-fold dilution steps separating homologous from heterologous precipitin titers. Results obtained with several tobamo- and tombusviruses indicated that the indirect form of the enzyme-linked immunosorbent assay (ELISA) can also be used for calculating SDI values. This was achieved by comparing the antiserum dilutions that lead to the same absorbance measurements (for instance 1.0) when homologous and heterologous viruses are assayed by ELISA. SDI values calculated from ELISA were similar to those obtained from precipitin tests. Because of its greater sensitivity, ELISA is able to quantify weak cross-reactions that are not detectable by precipitin tests.

The antigenic determinants or epitopes of a protein correspond to those parts of the molecule that are specifically recognized by the binding sites or paratopes of certain immunoglobulin molecules. Epitopes are thus relational entities that require complementary paratopes for their operational recognition. Some authors consider that the concept of epitope necessarily involves the two properties of antigenic reactivity (ability to bind to a paratope) and immunogenicity (ability to induce an immune response). Such a view creates difficulties because it makes the existence of epitopes in a protein depend on immunogenetic and regulatory mechanisms of the immunized host. The delineation of epitopes can be achieved by antigenic cross-reactivity studies or by X-ray crystallography. Both approaches require specific criteria for deciding which residues of the antigen are in contact with the paratope and are functionally part of the epitope. The relative contribution of static accessibility, segmental mobility and induced fit to immune recognition remains controversial. Each of the methods used for analysing antigenic specificity is subject to various operational constraints originating from the type of experimental probe and from the form at, sensitivity and specificity of the immunoassay used. If a protein is assumed to contain as many epitopes as the number of different monoclonal antibodies that can be raised against it, the delineation of epitopes corresponds to the summation in various hosts of the immune repertoire specific for the antigen. Neutralization epitopes are a special subclass of the epitopes of infectious agents and toxins that are specifically recognized by antibody molecules able to neutralize the biological activity of the antigen. The identification of neutralization epitopes is important for the development of synthetic vaccines because it is this type of epitope that should be mimicked by synthesis and used as a vaccine for eliciting protective immunity. The first demonstration that synthetic peptides could elicit antibodies that neutralized viral infectivity was made by Anderer and his colleagues in the 1960s in their work with tobacco mosaic virus. Nearly 20 years passed before it was shown that antibodies to synthetic peptides were also able to neutralize the infectivity of other viruses such as foot-and-mouth disease, polio and hepatitis B viruses.

Five monoclonal antibodies (McAbs) were raised to the tobamovirus, odontoglossum ringspot virus (ORSV). All five McAbs reacted with the virus in double antibody sandwich (DAS) ELISA but not in an ELISA using virus-coated plates. All the McAbs recognized a panel of ORSV strains and isolates, although one of the antibodies reacted better with some isolates and another reacted less with certain isolates than with type ORSV. It was possible to use the same McAbs both as coating and as biotinylated antibody in DAS-ELISA. None of the five McAbs was able to bind to orchid strains of tobacco mosaic virus (TMV). In order to detect strains of both viruses, ORSV and TMV, in infected orchids it was necessary to include also McAbs raised against TMV in the immunoassays. The use of a mixed polyclonal-monoclonal antibody DAS-ELISA system is advocated for detecting both tobamoviruses in orchids.

An enzyme-linked immunosorbent assay (ELISA) has been developed which allows hybridoma cell cultures to be tested for the presence of monoclonal antibodies specific for cyclosporin A. Immunization of mice wit free cyclosporin was found to be preferable to immunization with cyclosoporin-carrier conjugates. It is hoped that the availability of monoclonal antibodies to cyclosporin will clarify the contribution of cyclosporin metabolites to immunosuppression and nephrotoxicity.

The ability of cyclophilin to react with derivatives of cyclosporine (CsA) was studied. Cyclophilin was found to interact preferentially with CsA-residues 1, 2, 10 and 11, which, together with residue 3, are the residues known to contribute to the immunosuppressive activity of CsA. The recognition of different CsA-derivatives by cyclophilin was correlated with their immunosuppressive activity in vitro. All CsA-derivatives showing a significant activity did bind to cyclophilin, although some of the CsA-derivatives able to bind cyclophilin exhibited only low activities. The results suggest that binding to cyclophilin might be one requirement for immunosuppressive activity of CsA derivatives. When tested with CsA-derivatives showing various conformational changes, the binding of cyclophilin was strongly specific for the peptide-ring conformation of CsA. No binding of calmodulin to CsA could be detected in several formats of solid-phase enzyme- or radioimmunoassay, suggesting that, in contrast to cyclophilin, calmodulin does not possess sufficient affinity for CsA to bind to it when immobilized on the solid phase.