Epistemic_Statements  

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Id Subject Object Predicate Lexical cue
T1 247-412 Epistemic_statement denotes The basic property of a monoclonal antibody, that of combining specifically with one epitope (or family of related epitopes), can be used to supply a wealth of data.
T2 413-609 Epistemic_statement denotes A single monoclonal antibody can provide information on protein "relatedness," structure, function, synthesis, processing, cellular or tissue distribution and on the association between molecules.
T3 777-874 Epistemic_statement denotes However, it is appropriate to differentiate between the broad areas of virological investigation.
T4 875-955 Epistemic_statement denotes The contribution of monoclonal antibodies within each area can then be assessed.
T5 956-1223 Epistemic_statement denotes Thus, diagnostic virology, taxonomy, and epidemiology (Section 11) can be considered separately from the biochemical and molecular biological investigations described in Section 111; Section IV deals with the interaction of virus and host at the whole organism level.
T6 1437-1646 Epistemic_statement denotes It is not intended in this review to provide an exhaustive list of all publications in which monoclonal antibodies have been used, but rather to illustrate how monoclonal antibodies can be applied in virology.
T7 1837-2030 Epistemic_statement denotes It is important to emphasize that monoclonal antibody techniques are rarely applied in isolation, but are normally backed up with investigations using the more classical biochemical approaches.
T8 2570-2906 Epistemic_statement denotes Furthermore, there are limitations to the information that antibody studies can provide; structural studies for instance ultimately depend on the orientation of an antibody binding site on the various structural levels of the protein, amino acid sequence, three-dimensional structure, and even intermolecular associations (Section 111).
T9 3093-3149 Epistemic_statement denotes This has been recently reviewed (Gerhard et al., 1980) .
T10 3150-3309 Epistemic_statement denotes It is, however, necessary to consider the production of the immunogen, since it may be necessary to enrich for, or preferentially expose, a particular antigen.
T11 4032-4256 Epistemic_statement denotes Also, if cross reacting, e.g., group specific antibodies are required, it is possible to perform the primary inoculation with one antigen, and the boosting inoculation with the cross reacting antigen (Gerhard et al., 1978) .
T12 4472-4581 Epistemic_statement denotes However, it is relevant to consider the limitations of these processes when applied to monoclonal antibodies.
T13 4700-4781 Epistemic_statement denotes This may be related to the fact that only one epitope is recognized per molecule.
T14 4782-4874 Epistemic_statement denotes Consequently, extensive cross linking and stabilization of immune complexes is not possible.
T15 5156-5223 Epistemic_statement denotes This problem may be diminished in the case of a multimeric protein.
T16 5402-5483 Epistemic_statement denotes However, this detergent will have some destabilizing effect on protein structure.
T17 5484-5631 Epistemic_statement denotes Monoclonal antibodies are often sensitive to minor conformational change (Section II1,B) and consequently may be rendered nonreactive in this test.
T18 5632-5751 Epistemic_statement denotes It may be necessary to experiment with detergent type and concentration in order to achieve satisfactory precipitation.
T19 5854-6118 Epistemic_statement denotes The adverse effect of alterations in protein conformation may be aggravated in western blot procedures where antigens are often totally denatured for separation on sodium dodecyl sulfate (SDS)polyacrylamide gels before testing with antibody (Towbin et al., 1979) .
T20 6119-6205 Epistemic_statement denotes However, the method does have applications (Braun et al., 1983; Roseto et al., 1983) .
T21 6735-6979 Epistemic_statement denotes Detergent conditions are frequently milder than those used in immunoprecipitation, but the attachment of antigen to the solid support necessary in many cases for these tests can itself result in conformational alteration (Bruck et al., 1982a) .
T22 7228-7391 Epistemic_statement denotes These techniques have been widely used in diagnostic virology (Section II,A) as well as in studies of viral protein synthesis and virus maturation (Section 111,B).
T23 7591-7765 Epistemic_statement denotes In the following it is shown how application of these techniques in experiments utilizing monoclonal antibodies can be made in the study of all areas of virological research.
T24 7950-8235 Epistemic_statement denotes Although any one antibody is elicited by the exposure of an epitope to the immune system, it will react with many other epitopes to a greater or lesser extent, depending on the degree of fit between the would-be target site on the antigen and the combining site on the antibody itself.
T25 9848-10014 Epistemic_statement denotes Indeed this approach greatly exceeds conventional serology in specificity, and has led to the recognition of differences between viruses previously thought identical.
T26 10595-10791 Epistemic_statement denotes These investigations could lead to more effective vaccination programs or disease treatment, and to a greater understanding of the relationships between viruses, their evolution, and epidemiology.
T27 11039-11233 Epistemic_statement denotes High affinity for antigen, both to permit efficient combination with low concentrations of antigen and to more efficiently displace host antibodies which may already coat primary biopsy samples.
T28 11237-11378 Epistemic_statement denotes It may be directed toward an area of the molecule not normally recognized efficiently by the host's own antibodies, and not therefore masked.
T29 11382-11517 Epistemic_statement denotes The antibody should be directed against an often repeated antigenic determinant that is readily accessible, e.g., a virus coat protein.
T30 11521-11608 Epistemic_statement denotes Multivalence: this may increase sensitivity, and IgM was found more effective than IgG.
T31 11609-11849 Epistemic_statement denotes If the antibody is not carefully chosen, it may give less satisfactory results than usual serological procedures (Phillips et al., 19821 , and cross reaction with host cell polypeptides should be carefully excluded (Fujinami et al., 1983) .
T32 11850-12064 Epistemic_statement denotes The application of monoclonal antibodies in diagnosis can be illustrated by reference to the following viruses; herpes, hepatitis B, rabies, flaviviruses, picornaviruses, and influenza; this list is not exhaustive.
T33 13069-13410 Epistemic_statement denotes A similar procedure has also proved successful in the identification of varicella zoster (human herpesvirus 3) in cells obtained from vesicular skin lesions (Forghani et al., 1982) ' and monoclonal antibodies are now available which could be useful for typing Epstein -Barr virus (human gamma herpesvirus 4) (Mueller-Lantzsch et al., 1981) .
T34 13411-13517 Epistemic_statement denotes Herpes viruses can also be distinguished by a rapid enzyme immune filtration test (Richman et al., 1982) .
T35 13518-13686 Epistemic_statement denotes Marek's disease (MD) virus is a herpes virus of chickens normally diagnosed by reference antisera directed against the Marek's associated tumor surface antigen (MATSA).
T36 13864-14011 Epistemic_statement denotes Use of this test should help prevent confusion with lymphoid leukosis, since both diseases produce similar symptoms and are of economic importance.
T37 14582-14795 Epistemic_statement denotes All four serotypes of Dengue virus can be distinguished by monoclonal antibodies and have been applied with success to the identification of fresh low passage, virus isolates in an IF test (Henchal et al., 1983) .
T38 14880-15072 Epistemic_statement denotes Influenza A and B viruses could be readily distinguished and, in some cases, typed satisfactorily even when there was insufficient virus present to permit hemagglutination inhibition testing .
T39 15073-15174 Epistemic_statement denotes Finally monoclonal antibodies may find widespread future use in the diagnosis of plant virus disease.
T40 15175-15376 Epistemic_statement denotes Recent work with antibodies specific for tobacco mosaic virus was able to distinguish two orchid strains which were previously thought identical with the tabomavirus type strain (Briand et al., 1982) .
T41 15879-16019 Epistemic_statement denotes This confirmed the required similarity between vaccines in use and these strains could also be differentiated from virulent field isolates .
T42 16756-16847 Epistemic_statement denotes This may have an important bearing on instances in which vaccine failure has been observed.
T43 16848-16956 Epistemic_statement denotes Variation in the nucleocapsid protein is probably not so important in this respect (Charlton et al., 1982) .
T44 17554-17680 Epistemic_statement denotes The three-dimensional structure has been determined for both the hemagglutinin (HA) and neuraminidase (NA) ; Section III,A,3).
T45 17681-17767 Epistemic_statement denotes The maintenance of influenza within the human population is related to two mechanisms.
T46 17960-18034 Epistemic_statement denotes Preexisting immunity is thus overcome and a worldwide pandemic may result.
T47 18035-18214 Epistemic_statement denotes In the periods between antigenic shift, a more gradual alteration occurs in the virus proteins (antigenic drift) which in some cases may permit reinfection of the host population.
T48 18215-18473 Epistemic_statement denotes Monoclonal antibodies have naturally been applied to these most interesting phenomena in an attempt to demonstrate the relationships between human, animal, and avian virus species, and also to demonstrate antigenic drift both in the field and the laboratory.
T49 18720-18894 Epistemic_statement denotes A close relationship was noted between hemagglutinin molecules from those sources suggesting avian viruses might have played a role in the evolution of the seal virus strain.
T50 18895-19140 Epistemic_statement denotes In other studies differences have been noted between 25 avian (H4) influenza strains which may be related to the original host (Fukushi et al., 1982) and differences have been observed between neuraminidase (NA) molecules (Holmes et al., 1982) .
T51 19412-19510 Epistemic_statement denotes This demonstrates how such a process might occur under immunological pressure applied by the host.
T52 19511-19749 Epistemic_statement denotes At least some of these laboratory selected variants might have been able to spread in the population since they were not neutralized by many samples of sera from potential hosts which did recognize the parent virus (Natali et al., 1981) .
T53 20044-20114 Epistemic_statement denotes At least two variants of influenza B could coexist in the population .
T54 20115-20285 Epistemic_statement denotes The respective epidemiologies of herpes simplex virus 1 and 2 have also been studied and one patient may suffer from both viruses simultaneously (Peterson et al., 1983) .
T55 20819-20950 Epistemic_statement denotes This type of analysis could also indicate polypeptides which have been structurally conserved during the course of virus evolution.
T56 21049-21123 Epistemic_statement denotes However, it is essential that the monoclonal antibody is chosen with care.
T57 21124-21305 Epistemic_statement denotes Many will recognize carbohydrate determinants of glycosylated polypeptides, and this modification is influenced by the host cell in which the virus is grown (Klenk and Rott, 1980) .
T58 21584-21815 Epistemic_statement denotes Binding of monoclonal antibody to antigen may or may not disrupt the biological function of that protein; this could lead to errors if the tests involved monitor antibody binding indirectly by inhibition of target protein function.
T59 22182-22469 Epistemic_statement denotes However, binding alone is an insufficient guide for research purposes, since the functional significance of the same epitope may vary with the virus strain and mutations may render an antibody unable to inhibit protein function without abolishing antibody binding (Kendal et al., 1981) .
T60 22470-22578 Epistemic_statement denotes It is necessary therefore to perform a number of tests in order to extract the maximum possible information.
T61 22579-22724 Epistemic_statement denotes Monoclonal antibodies provide us with a tool which is able to examine the individual protein within a structure as complicated as the whole cell.
T62 22725-22805 Epistemic_statement denotes It is possible to investigate, both in uitro and in viuo, the role of a protein.
T63 22806-22941 Epistemic_statement denotes It is also possible to determine the active areas of the molecule and to monitor its synthesis and processing within the infected cell.
T64 23089-23194 Epistemic_statement denotes The combination of antibody with antigen can be used to provide structural information on several levels.
T65 23195-23426 Epistemic_statement denotes First, just as it is possible to identify proteins carrying a taxonomic marker, so it is possible to identify a protein as performing a particular function once an antibody is available which is capable of inhibiting that function.
T66 23708-23771 Epistemic_statement denotes In this way epitopes may be assigned a functional significance.
T67 23772-23981 Epistemic_statement denotes If certain assumptions are made, it is then possible to conduct competitive binding analyses to arrange the antibody sites relative to each other, and a schematic map of the molecule's surface can be drawn up.
T68 23982-24069 Epistemic_statement denotes Frequently virus variants can be selected to which a given antibody can no longer bind.
T69 24070-24182 Epistemic_statement denotes These may also be analyzed for loss of protein function or loss of capacity to bind other monoclonal antibodies.
T70 24183-24250 Epistemic_statement denotes In this way the map can be refined, or indeed constructed entirely.
T71 24404-24686 Epistemic_statement denotes This can be done by determination of the amino acid alterations produced in the variants described above, either directly by partial proteolysis and examination of those molecule fragments bound by the antibody, or from genome sequence determination in the region of the alteration.
T72 24687-24795 Epistemic_statement denotes Active amino acid residues can thus be distinguished and positioned in the primary structure of the protein.
T73 24796-24986 Epistemic_statement denotes The location of the antibody binding site is then known if the three-dimensional structure of the protein has been determined, or clues may be obtained from immune electron microscopy (IEM).
T74 25350-25654 Epistemic_statement denotes In this manner, those proteins to which neutralizing antibodies are directed have been determined, e.g., VP1 in poliovirus (Osterhaus et al., 1981b; Thorpe et al., 1982) , VP2 in Bluetongue virus Letchworth and Appleton, 1983) , and gp 56 in Venezuelan equine encephalitis virus (Roehrig et at., 1982a) .
T75 25990-26176 Epistemic_statement denotes The antibody used in this work was apparently capable of distinguishing between T antigen with this activity and T antigen which could not perform this function (Scheller et al., 1982) .
T76 26534-26715 Epistemic_statement denotes Although the last two experiments are concerned with the function of cellular proteins, this approach has an obvious application in the study of virus replication and transcription.
T77 26716-26814 Epistemic_statement denotes Such an approach could also investigate the involvement of host -cell proteins in these processes.
T78 27021-27229 Epistemic_statement denotes This protein was detected in cancer effusions and found to be highly efficient at blocking monocyte response to chemoattractants, a function which could protect a cancer from attack (Cianciolo et al., 1981) .
T79 27562-27654 Epistemic_statement denotes In principle, the same approach could be extended to any protein with a measurable activity.
T80 27655-27760 Epistemic_statement denotes Combination of antibody with antigen can have a variety of effects, de-pending on the site of attachment.
T81 27761-27908 Epistemic_statement denotes Some functions which might previously have been thought associated, have been found separable when highly specific monoclonal antibodies were used.
T82 27909-28236 Epistemic_statement denotes A polyclonal monospecific serum which coats the protein surface with antibody will normally inhibit all protein functions, but a monoclonal antibody, which binds away from the active site need not have any detectable effect at all (Burstin et al., 1982; Massey and Schochetman, 1981; Pinter et al., 1982; Thorpe et al., 1982) .
T83 29018-29094 Epistemic_statement denotes This indicates that the requirements underlying these reactions must differ.
T84 29095-29428 Epistemic_statement denotes Both of these tests in fact are artificial, since the virus does not normally act to cross link cells, or to lyse the cell it is about to attack, and these results indicate that it is possible to prevent stable cell cross linking in circumstances which still permit sufficient virus/cell association for action of the fusion protein.
T85 29429-29688 Epistemic_statement denotes Similarly, antibodies may inhibit hemagglutination but still permit sufficient cell contact such that infection is not inhibited (Burstin et al., 1982; Gentry et al., 1982; Gonzalez-Scarano et al., 1982; Kimura-Kiroda and Yasui, 1983; ter Meulen et al., 1981.
T86 29689-29794 Epistemic_statement denotes This type of analysis provides clues that the functional areas of a multifunctional protein are distinct.
T87 29892-30181 Epistemic_statement denotes These probably reside at different areas of the molecule since some monoclonal antibodies inhibit only neuraminidase (Orvell and Grandien, 1982) , and virus variants have been obtained under monoclonal antibody selection pressure which are deficient only in neuraminidase (Portner, 1981) .
T88 30278-30370 Epistemic_statement denotes Presumably these antibodies bind to sites important in virus penetration but not absorption.
T89 30536-30709 Epistemic_statement denotes Other biological functions have been used to characterize epitopes defined by competition ELISA on the envelope glycoprotein of bovine leukemia virus (Bruck et al., 1982a) .
T90 30896-31134 Epistemic_statement denotes Monoclonal antibodies directed to virus surface proteins are generally more readily produced, but as more become available specific for virus internal proteins, this approach can be expected to yield further information on their function.
T91 31135-31444 Epistemic_statement denotes A function inhibihon assay has been applied to delineate that area of the SV40 T antigen involved in ATPase activity (Clark et d., 1981) and to demonstrate that some areas of the influenza and VSV nucleocapsid proteins may be associated with the transcription process (De et al., 1982; van Wyke et d., 1981) .
T92 31580-31706 Epistemic_statement denotes This is conveniently achieved by a competitive binding analysis although selection of variants has also been extensively used.
T93 32033-32151 Epistemic_statement denotes However, several precautions must be taken in the interpretation of data before meaningful conclusions can be reached.
T94 32397-32463 Epistemic_statement denotes However, this is not the only explanation for such an observation.
T95 32464-32623 Epistemic_statement denotes Binding sites may be distinct but close together, such that the bulk of the first antibody molecule sterically prevents access of a second to its binding site.
T96 32624-32737 Epistemic_statement denotes These two possibilities cannot be readily distinguished but do at least both imply proximity of attachment sites.
T97 32738-32921 Epistemic_statement denotes In fact, a monoclonal antibody bound to a single epitope on its target protein may have considerable "swivel" movement available to it rendering steric effects relatively unimportant.
T98 32922-33046 Epistemic_statement denotes Stone and Nowinski (1980) reported that two IgG molecules (MW 150K) could bind noncompetitively to a retrovirus 15K protein.
T99 33047-33185 Epistemic_statement denotes However, immunoglobulin class may well be important here since oligomeric IgM would obviously provide a greater steric hindrance than IgG.
T100 33186-33312 Epistemic_statement denotes If steric effects are suspected they may be minimized by the use of monomeric antigen-binding submolecular antibody fragments.
T101 33313-33457 Epistemic_statement denotes It is however quite possible that allosteric inhibitory effects might also occur which would be indistinguishable from true competitive binding.
T102 33458-33628 Epistemic_statement denotes Indeed positive allosteric effects have been observed and antibody pairs identified which bind synergistically (Lefrancois and Lyles, 1982a,b; Lubeck and Gerhard, 1982) .
T103 33629-33721 Epistemic_statement denotes Similar effects resulting in competition may be widespread but are difficult to demonstrate.
T104 33722-33961 Epistemic_statement denotes Combination of monoclonal antibody with influenza neuraminidase modifies the reaction kinetics (Mountford et al., 1982) and combination with HSV glycoprotein can alter the pattern obtained by partial proteolysis (Eisenberg et al., 1982a) .
T105 33962-34041 Epistemic_statement denotes These effects could also be mediated by antibody-induced conformational change.
T106 34172-34345 Epistemic_statement denotes Taking an extreme case, a weakly binding antibody would not show detectable competition with a labeled, strongly binding immunoglobulin, but the reverse is clearly not true.
T107 34346-34492 Epistemic_statement denotes In practice a spectrum of relative interference efficiencies will probably be obtained (Lefrancois and Lyles, 1982a,b; Stone and Nowinski, 1980) .
T108 34619-34785 Epistemic_statement denotes For this reason binding sites should not be distinguished on the basis of the extent of competition, it is the observation of competition itself which is significant.
T109 34967-35142 Epistemic_statement denotes Any such noncompeting antibodies should be examined in the reverse experiment (i.e., with the other partner carrying the marker) to exclude avidity effects as described above.
T110 35143-35246 Epistemic_statement denotes If this procedure fails to reveal competition, then allocation to separate binding groups is justified.
T111 35247-35474 Epistemic_statement denotes Alternatively, determination of antibody avidity may obviate the need for this analysis but one-way competition effects can still occur even in the case of antibodies of similar avidity and concentration (Carter et al., 1982) .
T112 35475-35557 Epistemic_statement denotes The reasons for this are unknown but might involve some form of allosteric effect.
T113 35558-35804 Epistemic_statement denotes Determination of antibody avidity may be performed by a rapid method (Jackson et al., 1983) or relative antibody avidities may be estimated from RIA saturation curves (Carter et al., 1982; Massey and Schochetman, 1981; Stone and Nowinski, 1980) .
T114 36772-36979 Epistemic_statement denotes The manner in which the sample is prepared can also influence the conformation and/or exposure of the reacting epitopes and this should be considered in the design of such experiments (Bruck et al., 1982b) .
T115 36980-37138 Epistemic_statement denotes The second approach to the problem of arranging binding sites relative to each other is to select virus variants under monoclonal antibody selection pressure.
T116 37214-37323 Epistemic_statement denotes Nonneutralizing antibodies could also be applied to enveloped viruses if used in conjunction with complement.
T117 37324-37483 Epistemic_statement denotes Virus variants may then be isolated to which the selecting antibodies can no longer bind, or in which antibody combination no longer results in neutralization.
T118 37631-37682 Epistemic_statement denotes In this way, a map of epitopes can be constructed .
T119 38072-38204 Epistemic_statement denotes This type of experiment provides a finer analysis of the relationships between binding sites because steric hindrance is eliminated.
T120 38443-38669 Epistemic_statement denotes However, regardless of the method in which epitope topography is determined, the schematic map thus produced must be oriented on the physical structure of the protein and this necessitates a detailed knowledge of the molecule.
T121 38670-38726 Epistemic_statement denotes Two approaches have been used to achieve this objective.
T122 39250-39336 Epistemic_statement denotes In this way a linear proteolytic leavage map could be derived for 8 different enzymes.
T123 39337-39530 Epistemic_statement denotes Monoclonal antibodies were then analyzed for their ability to immunoprecipitate various polypeptide fragments and their binding sites could then be assigned in the molecule's primary structure.
T124 39531-39689 Epistemic_statement denotes It was found that antibody binding sites were closely grouped in two areas which presumably reflected regions of the protein exposed for immunological attack.
T125 40554-40706 Epistemic_statement denotes Provided the primary sequence of the molecule is known, virus variants can supply highly specific information on the location of antibody binding sites.
T126 40707-40889 Epistemic_statement denotes Unlike antibody binding to a peptide fragment, however, it is not possible to be absolutely certain that the amino acid affected actually constitutes part of the binding site itself.
T127 40890-40957 Epistemic_statement denotes This method has been extensively applied in the study of influenza.
T128 40958-41102 Epistemic_statement denotes Amino acid alterations can be detected by peptide mapping (Laver et al., 1979 (Laver et al., , 1980b or genome sequencing (Caton et al., 1982) .
T129 41103-41234 Epistemic_statement denotes Since the three-dimensional structure of the influenza hemagglutinin is now available these binding sites could be easily located .
T130 41827-42024 Epistemic_statement denotes This also provided insight into those areas of the molecule involved in enzyme function and previously identified by monoclonal antibodies as well as locations in which antigenic drift could occur.
T131 42233-42398 Epistemic_statement denotes This was found to be a small (eight amino acid) sequence in protein VP1 Minor et al., 1983) ; however no three-dimensional structure is yet available for this virus.
T132 42399-42626 Epistemic_statement denotes A further clue to antibody binding site location may be obtained from immune electron microscopy in which the antibody is used in conjunction with an electron-dense material such as ferritin or hemocyanin (Gonda et al., 1981) .
T133 42854-43077 Epistemic_statement denotes A similar situation has also been demonstrated in influenza and may explain the separation of hemagglutination inhibition and neutralization exhibited by monoclonal antibodies specific for the HA protein of seal influenza .
T134 43299-43367 Epistemic_statement denotes In this way they can answer questions of molecular and cell biology.
T135 43368-43553 Epistemic_statement denotes They can thus provide information concerning temporal and spatial separation of protein formation and accumulation, and data on protein modification and processing in the infected cell.
T136 43826-43968 Epistemic_statement denotes Rather we shall consider persistent infections where normal protein synthesis is modified or controlled such that virus and cells may coexist.
T137 43969-44199 Epistemic_statement denotes Interesting information has been gathered in this area which frequently involves low levels of infected cells or proteins within those cells and monoclonal antibodies are thus admirably suited for investigation of this phenomenon.
T138 44200-44391 Epistemic_statement denotes Measles virus is associated with a persistent slowly progressing fatal infection of the human central nervous system (CNS) termed subacute sclerosing panencephalitis (SSPE) (reviewed by ter .
T139 44392-44541 Epistemic_statement denotes This disease is associated with a failure to produce mature virus or to develop the typical measles virus cytopathology, cell fusion in brain tissue.
T140 45199-45370 Epistemic_statement denotes Although this method did permit identification of M protein in a laboratory-established persistent infection which was previously thought not to express this polypeptide .
T141 45371-45483 Epistemic_statement denotes Monoclonal antibodies thus provide excellent evidence that matrix protein is not synthesized in SSPE cell lines.
T142 45484-45639 Epistemic_statement denotes The lack of this major structural polypeptide is thought to prevent virus particle maturation and account for the slowly progressing nature of the disease.
T143 46254-46384 Epistemic_statement denotes Perhaps this is one way in which persistently infected cells evade the immune system and thus permit the maintenance of infection.
T144 47159-47276 Epistemic_statement denotes In addition, a tumor antigen of adenovirus 5 has been shown to accumulate in the cell nucleus (Sarnow et al., 1982) .
T145 47384-47632 Epistemic_statement denotes This property is particularly relevant where precursorproduct relationships exist (as in proteolysis), or where similar sequences of proteins might occur in different molecules, e.g., following a recombination or readthrough event in transcription.
T146 47633-47807 Epistemic_statement denotes If the precursors never reach appreciable levels within the cell or the shared sequences are small, the cross reacting proteins are difficult to detect with polyclonal serum.
T147 48363-48484 Epistemic_statement denotes This type of analysis defines aprotein family but does not indicate whether the submolecular fragments serve any purpose.
T148 48485-48651 Epistemic_statement denotes Furthermore, care should always be taken to ensure that the protein precipitation is not caused by a spurious cross reaction with a host cell polypeptide (Section V).
T149 49568-49742 Epistemic_statement denotes Peptide mapping analysis showed that it apparently contained all the nucleocapsid peptides plus some new ones whose origin could not be determined (Wild and Giraudon, 1982) .
T150 49855-49941 Epistemic_statement denotes The origin of this protein is still obscure but a readthrough mechanism was suggested.
T151 50270-50353 Epistemic_statement denotes Following its synthesis, a protein may mature through posttranslational processing.
T152 50354-50589 Epistemic_statement denotes Processing in the sense used here includes covalent modification through glycosylation, posttranslational cleavage etc, as well as maturation by conformational changes often associated with the formation of intermolecular associations.
T153 50746-50918 Epistemic_statement denotes Conformational change is probably involved in this process and may perform a regulatory function as in tobacco mosaic virus assembly (Butler, 1971; Klug and Durham, 1971 ).
T154 51065-51223 Epistemic_statement denotes These proteins are degraded only in some cell types (Pereira et al., ,1982a which suggests this process is not a functionally important processing phenomenon.
T155 51224-51484 Epistemic_statement denotes However, monoclonal antibodies have been used in pulse-chase experiments to demonstrate genuine modification of proteins through glycosylation (Balachandran et al., 1981) and also to demonstrate temporal regulation of their synthesis (Showalter et al., 1981) .
T156 51704-52063 Epistemic_statement denotes However, monoclonal antibodies may not react with the unmodified form of a protein (only one antibody from a panel of 65 directed against measles virus H protein reacted with the unmodified product formed in vitro; M. J. Carter, unpublished observation) and consequently might not always provide an adequate probe for the expression of the polypeptide moiety.
T157 53048-53139 Epistemic_statement denotes This association between a gag and an env gene product may be important for virus assembly.
T158 53228-53338 Epistemic_statement denotes It is likely that protein combination to form virus structural components will involve conformational changes.
T159 53339-53425 Epistemic_statement denotes Protein conformation could also be altered artifactually during extraction procedures.
T160 53426-53711 Epistemic_statement denotes Although some antibodies seem to recognize a sequence of amino acids, most monoclonal antibodies possess a sensitivity to the conformation of the epitope that cannot be visualized using a polyclonal serum, and this permit investigations into this complex field of structural isomerism.
T161 53712-53916 Epistemic_statement denotes Alternatively the development of intermolecular associations must result in the masking of some epitopes and therefore the inhibition of combination with antibody directed toward that area of the protein.
T162 53917-54153 Epistemic_statement denotes Monoclonal antibodies could distinguish readily between native and unfolded forms of the Sindbis virus glycoprotein El (Roehrig et al., 1982b) and between native (N) and heated (H) forms of the poliovirus capsid Icenogle et al., 1981) .
T163 54154-54293 Epistemic_statement denotes In the latter case it was also shown that the irreversible conversion of N to H could be accomplished by some acidic isolation procedures .
T164 55128-55330 Epistemic_statement denotes However, variants could readily be selected which did not express these sites, so it is likely that the areas recognized by these antibodies are not vital for structural integrity (Emini et al., 1982) .
T165 55331-55571 Epistemic_statement denotes Similar data have been reported for foot and mouth disease virus (FMDV) where monoclonal antibodies have been obtained which can distinguish between complete (146s) virions and their precursor (12 S) subunits (McCullogh and Butcher, 1982) .
T166 55572-55816 Epistemic_statement denotes These two studies demonstrate that unique antigenic sites are created during picornavirus maturation and explain earlier reports that neutralizing antibodies could only be elicited by using the mature polio virion as immunogen (Dernick, 1981) .
T167 55817-56012 Epistemic_statement denotes A similar effect has been demonstrated in the case of TMV where some monoclonal antibodies react only with the nucleocapsid, and others only with the protein monomer (Dietzgen and Sander, 1982) .
T168 56013-56279 Epistemic_statement denotes Although the former class must recognize a new site caused by conformational change or formed at molecular junctions, the latter class of antibodies could be directed at a site which is either masked in the protein complex or destroyed through conformational change.
T169 56404-56564 Epistemic_statement denotes The monomeric or denatured hexon protein lacks antigenic determinants in common with the trimeric capsomere, a situation reminiscent of the picornavirus capsid.
T170 56974-57206 Epistemic_statement denotes A structural rearrangement may also occur during release of CMV since some antibodies are able to react with determinants and neutralize infectious virus but are unable to bind to the same proteins on the surface of infected cells .
T171 57207-57369 Epistemic_statement denotes Conformational changes are believed important in the development of fusion protein activity by the influenza virus hemagglutinin at low pH (Skehel et al., 1982) .
T172 57636-57824 Epistemic_statement denotes For instance gp 56 and gp 50 have been shown closely associated on the Venezuelan equine encephalitis (VEE) virus envelope by means of competitive binding studies (Roehrig et al., 1982a) .
T173 57982-58081 Epistemic_statement denotes A complex is not apparently formed since these antigens did not cocap (Hackett and Askonas, 1982) .
T174 58082-58303 Epistemic_statement denotes On the mature influenza virus envelope however, the HA protein is often found in close association with the NA protein, since monoclonal antibodies specific for hemagglutinin may have a neuraminidase inhibiting activity .
T175 58304-58536 Epistemic_statement denotes Frequently, virus-specific products associate with cellular proteins and this can be demonstrated by competitive binding assays or coprecipitation experiments (Carroll and Gurney, 1982; Ferracini et al., 1982; Sarnow et al., 1982) .
T176 58551-58639 Epistemic_statement denotes Structural change can also result from the acquisition of mutations by the virus genome.
T177 59565-59772 Epistemic_statement denotes An examination based solely on radioimmune precipitation may be superior in detecting certain changes which "loosen" the protein and render it more susceptible to the action of detergents in the buffer used.
T178 59773-59948 Epistemic_statement denotes However, changes in relative avidity, topography, or allostery would not be detected provided antibody avidity was still sufficiently high to permit precipitation (Section I).
T179 59949-60038 Epistemic_statement denotes Similarly, detergent produced effects might not be detected in the competition RIA alone.
T180 60241-60379 Epistemic_statement denotes One obvious example of the use to which monoclonal antibodies can be put is in the preparation of affinity columns for such purifications.
T181 60380-60583 Epistemic_statement denotes It is not appropriate to list here all instances in which this has been performed, but any review of the application of monoclonal antibody technology would be incomplete without mention of this subject.
T182 61093-61130 Epistemic_statement denotes Monoclonal antibodies may be used to
T183 61131-61223 Epistemic_statement denotes The isolation of nonneutralizable virus variants has already been discussed (Section II1,A).
T184 61224-61316 Epistemic_statement denotes These variants can also be applied in studies of pathogenicity as well as protein structure.
T185 61317-61521 Epistemic_statement denotes Spontaneous virus variants occur at a high frequency (lo-*) in Coxsackie B4 virus populations (Prabhaker et al., 1982) and this may explain the high variation in disease pattern produced by these viruses.
T186 61522-61617 Epistemic_statement denotes Possibly alterations in the virus attachment protein might result in an altered tissue tropism.
T187 61618-61716 Epistemic_statement denotes However, the most interesting data have so far emerged from studies of rabies and influenza virus.
T188 62094-62332 Epistemic_statement denotes These variants were very similar to the parental virus by all other criteria, but differed in their capacity to evoke a strong and rapid immune response which could lead to successful defense against the infection (Coulon et al., 1982~) .
T189 63456-63667 Epistemic_statement denotes Although antigenic drift has been studied mainly in the HA molecule (Natali et al., 1981; Underwood, 1982; it may also occur in the neuraminidase but its extent may be restricted in some species of avian virus .
T190 63988-64138 Epistemic_statement denotes Monoclonal antibodies can be applied in order to mimic the immune response and to gain insight into the effect of immunological pressure on the virus.
T191 64139-64428 Epistemic_statement denotes Experiments using monoclonal antibodies to block cytotoxic lymphocyte action have demonstrated that the epitopes recognized by these cells may be identical to (Finberg et al., 1982) or distinct from (Lefrancois and Lyles, 1983b) those regions to which neutralizing antibodies are directed.
T192 64429-64538 Epistemic_statement denotes The host immune response has been implicated in the maintenance of measles virus persistence leading to SSPE.
T193 64539-64647 Epistemic_statement denotes As described in Section JII,B, the disease is associated with a defect in the production of virus M protein.
T194 64648-64869 Epistemic_statement denotes Some evidence has been gathered that suggests the hosts' immune response itself may actually modulate the synthesis of virus polypeptides and consequently help to bring this situation about (Fujinami and Oldstone, 1979 ).
T195 64870-65094 Epistemic_statement denotes This has been modelled in uiuo where inoculation of a hyperimmune anti-measles virus antiserum was found to alter the characteristics of disease in intracerebrally inoculated BALB/c mice, a delayed disease was then produced.
T196 65095-65317 Epistemic_statement denotes Inoculation of monoclonal antibody specific for the virus hemagglutinin protein was also able to induce this effect although monoclonal antibody directed against the nucleocapsid protein could not (Rammohan et al., 1981) .
T197 65580-65694 Epistemic_statement denotes Once again monoclonal antibody to H, but not N could produce this effect (P. N. Barrett, personal communication) .
T198 65695-65806 Epistemic_statement denotes Antibody to surface glycoprotein can also prolong experimental Sindbis virus infection (Chanas et al., 1982a) .
T199 66174-66279 Epistemic_statement denotes However, it is also true that under some circumstances monoclonal antibodies can enhance virus infection.
T200 66492-66619 Epistemic_statement denotes However, use of monoclonal antibodies has demonstrated that the virus epitope to which the antibody is bound is also important.
T201 66939-67018 Epistemic_statement denotes Use of monoclonal antibodies for therapeutic purposes is as yet in its infancy.
T202 67019-67086 Epistemic_statement denotes It is likely that the antibodies would be used in three main areas.
T203 67087-67175 Epistemic_statement denotes First, in emergency, injection of a neutralizing antibody can aid recovery from disease.
T204 67176-67345 Epistemic_statement denotes Human monoclonal antibodies are now becoming available (Crawford et al., 1983; Sikora and Neville, 1982) and are likely to produce fewer problems with allergic reaction.
T205 67346-67452 Epistemic_statement denotes Animal experiments in the mouse have indicated that monoclonal antibodies will be beneficial in this area.
T206 67973-68082 Epistemic_statement denotes This action was however dependent upon the breakdown of the blood-brain barrier (Doherty and Gerhard, 1981) .
T207 68083-68211 Epistemic_statement denotes The ability of antibody to function in this way is not simply correlated with its anti-virus action measured by in uitro assays.
T208 68212-68488 Epistemic_statement denotes Nonneutralizing antibodies protect mice from HSV (Balachandran et al., 1982c; Rector et al., 1982) or Sindbis virus (Schmaljohn et al., 1982) , and Mathews and Roehrig (1982) found nonneutralizing antibodies could also protect mice from Japanese equine encephalitis infection.
T209 68822-69032 Epistemic_statement denotes Some evidence suggests that complement is not involved in protection by monoclonal antibodies and suggests the process is mediated by an antibody-dependent cell-mediated cytolysis (Balachandran et al., 1982~) .
T210 69211-69318 Epistemic_statement denotes Heterogeneity among Coxsackie (Prabhakar et al., 1982) or bluetongue viruses ) may permit such an approach.
T211 69319-69424 Epistemic_statement denotes The antibodies could also be used in the preparation of pure subunit vaccines (Osterhaus et al., 1981b) .
T212 70122-70273 Epistemic_statement denotes As three-dimensional structures of more molecules are determined, so this work should extend our understanding of virus protein structure and function.
T213 70274-70423 Epistemic_statement denotes In the foregoing discussion it has been emphasized how the specificity of a monoclonal antibody can be applied to the study of all areas of virology.
T214 70424-70579 Epistemic_statement denotes In each case it is the specificity of the antibody which has proved useful, and even permitted the identification of related but hitherto unknown proteins.
T215 70580-70706 Epistemic_statement denotes However, this very specificity also provides the basis for spurious cross reactivity not normally present in polyclonal serum.
T216 70863-71020 Epistemic_statement denotes A single epitope repeated on another protein would cross react, but this would be masked by the vast majority of antibodies reacting with the target antigen.
T217 71021-71073 Epistemic_statement denotes In the case of a monoclonal antibody this is not so.
T218 71074-71278 Epistemic_statement denotes A target molecule may have only one site for the antibody and if this is shared with another protein a considerable cross reaction could be detected, even in the abscence of extensive structural homology.
T219 71279-71501 Epistemic_statement denotes It has been calculated (Crawford et al., 1982 ) that a primary sequence of four amino acids (a not unusual size for an antibody binding site) might occur some 15 times in the average cell's complement of protein sequences.
T220 71502-71902 Epistemic_statement denotes However, monoclonal antibody cross reactions would be expected much less frequently than this because (1) the amino acid sequence must be expressed on the surface of the molecule, (2) antibodies may recognize a sequence in a given conformation or state of modification, (3) protein sequence does not normally occur at random, and (4) antibody binding sites are often larger than this (Atassi, 1975) .
T221 71903-72084 Epistemic_statement denotes Furthermore, it would be very difficult for an animal to produce an antibody response to an epitope which was likely to cross react, merely on a random basis, with a "self" epitope.
T222 72085-72200 Epistemic_statement denotes However, this could perhaps be tolerated as a small constituent of a total non-self-cross-reacting immune response.
T223 72201-72285 Epistemic_statement denotes Consequently, it is likely that fortuitous cross reaction may be comparatively rare.
T224 72286-72416 Epistemic_statement denotes Nevertheless, many cross reactions have been reported which are at present not understood (reviewed by Lane and Koprowski, 1982) .
T225 72603-72829 Epistemic_statement denotes In the former case there is good reason to believe that some of these antibodies recognize epitopes on functionally related host proteins, and which therefore may be constrained to some degree of overall structural similarity.
T226 72830-72977 Epistemic_statement denotes Whether this represents an evolutionarily conserved structure, or one which has arisen by independent convergent evolutionary processes is unknown.
T227 72978-73158 Epistemic_statement denotes However, cross reaction at the monoclonal antibody level cannot be assumed indicative of any extensive structural or functional similarity without considerable supporting evidence.
T228 73371-73603 Epistemic_statement denotes In addition, the process described above provides an entirely new dimension to these procedures and might thus permit the identification of functionally related proteins which bear no evidence of cross reaction with polyclonal sera.
T229 73604-73813 Epistemic_statement denotes Consequently application of monoclonal antibodies has not only improved information obtained from well established assay procedures, but may also lead to processes yielding an entirely new type of information.