Anti-myeloperoxidase autoantibodies react with native but not denatured myeloperoxidase.

We wondered whether anti-myeloperoxidase (MPO) autoantibodies (MPO-ANCA) found in patients with systemic vasculitis react with a conformational epitope or epitopes on the MPO molecule. Sera from 15 human MPO-ANCA, and a polyclonal and a monoclonal anti-MPO antibodies were reacted with MPO in native and denatured states. Human MPO-ANCA and mouse monoclonal anti-MPO reacted with native MPO, and a 120-kD band representing the MPO hologenzyme, but not with denatured MPO fragments; however, MPO-ANCA and mouse anti-MPO did not demonstrate competitive inhibition of binding to MPO. Polyclonal rabbit anti-MPO reacted with both native and denatured MPO. All MPO-ANCA tested showed the same patterns of reactivity with native and denatured MPO in dot blot and Western blot analyses. Both polyclonal and monoclonal anti-MPO antibodies inhibited MPO's protein iodination by over 90%, whereas MPO-ANCA IgGs, normal IgGs and disease control IgGs did not. These data suggest that (i) MPO-ANCA interact with a conformational epitope on the MPO molecule; and (ii) MPO-ANCA from different patients have similar reactivity with native versus denatured MPO.     

Production of anti-dengue NS1 monoclonal antibodies by DNA immunization

Monoclonal antibodies against dengue NS1 protein were generated following immunization of mice with plasmid DNA encoding the transmembrane form of NS1 from dengue serotype 2 virus. A mammalian expression vector, pDisplay, was engineered to direct cell surface expression of dengue NS1 and tested for transient expression in COS cells. Two mice were immunized intramuscularly with six doses of 100 microg of plasmid at 2-week intervals; one mouse received a booster of live virus prior to the last plasmid injection. Both mice showed antibody responses against dengue antigens in dot enzyme immunoassay. Following fusion, hybridomas were screened with dot enzyme immunoassay against all four dengue serotypes. Specificity to the NS1 protein was confirmed by western blot analysis. Among five anti-dengue NS1 monoclonal antibodies generated, two clones were serotype 2 specific, two clones reacted with all four serotypes and the last also reacted with Japanese encephalitis virus. Reactivity against native or denatured forms of NS1 revealed three clones with reactivity to linear epitopes and two clones recognizing conformational epitopes. Such diverse specificity of anti-dengue NS1 monoclonal antibodies indicates that DNA immunization, especially with the combination of virus boosting, is an efficient way of producing monoclonal antibodies against viral protein. This has opened up a possibility of producing monoclonal antibodies to rare viral proteins that are difficult to isolate or purify.     

Epitopic mapping of linear and conformation-dependent antigenic determinants on GP5 of five U.S. bluetongue viruses.

Two distinct antigenic determinants of the major outer capsid protein, GP5, of five U.S. bluetongue viruses have been identified and mapped using monoclonal and oligoclonal antibodies. One antigenic site, identified by oligoclonal antibody AK-15, was found to be common and conserved in all five U.S. BTV serotypes. This linear epitope was located between amino acid residues 175 and 189 (ALQREAAERSEDEIK). The second determinant identified by monoclonal antibody 34.7 was present in BTV-2, -10, -11, and -17 but absent in BTV-13. The binding of this monoclonal antibody to GP5 could be blocked specifically by one of three short synthetic peptides located among amino acid residues 33-42 (KAAERFAESE), 159-168 (EKILKEEDSK), and 206-215 (EIERDGMQEE), indicating that this antigenic determinant is conformation-dependent. Oligoclonal antibody (AK-15) reacted with denatured GP5 immobilized on nitrocellulose membrane after Western transfer as well as with native GP5 present on the surface of purified BTV virions. Monoclonal antibody (34.7) reacted only with denatured GP5 but not native GP5 using an ELISA assay. However, these two antigenic epitopes alone did not elicit detectable neutralizing antibodies as determined by plaque reduction assay.     

Monoclonal antibodies to human chorionic gonadotropin and their application to two-site sandwich radioimmunoassay

Monoclonal antibodies were prepared against human chorionic gonadotropin (HCG). One monoclonal antibody recognized a conformational determinant expressed only on native HCG molecule and another monoclonal antibody had the specificity for the epitopes located on the beta-subunit of HCG. Monoclonal antibodies reacting with different antigenic determinants on the HCG molecule were used to develop a simplified 2-site sandwich radioimmunoassay in which one monoclonal antibody was immobilized and another labeled with 125iodine. This assay was highly specific for HCG and there was no cross-reactivity with alpha, beta-subunit of HCG, luteinizing hormone and follicle stimulating hormone.     

B-Cell Epitopes and Quantification of the ESAT-6 Protein of Mycobacterium tuberculosis

ESAT-6 is an important T-cell antigen recognized by protective T cells in animal models of infection with Mycobacterium tuberculosis. In an enzyme-linked immunosorbent assay (ELISA) with overlapping peptides spanning the sequence of ESAT-6, monoclonal antibody HYB76-8 reacted with two peptides in the N-terminal region of the molecule. Assays with synthetic truncated peptides allowed a precise mapping of the epitope to the residues EQQWNFAGIEAAA at positions 3 to 15. Hydrophilicity plots revealed one hydrophilic area at the N terminus and two additional areas further along the polypeptide chain. Antipeptide antibodies were generated by immunization with synthetic 8-mer peptides corresponding to these two regions coupled to keyhole limpet hemocyanin. Prolonged immunization with a 23-mer peptide (positions 40 to 62) resulted in the formation of antibodies reacting with the peptide as well as native ESAT-6. A double-antibody ELISA was then developed with monoclonal antibody HYB76-8 as a capture antibody, antigen for testing in the second layer, and antipeptide antibody in the third layer. The assay was suitable for quantification of ESAT-6 in M. tuberculosis antigen preparations, showing no reactivity with M. bovis BCG Tokyo culture fluid, used as a negative control, or with MPT64 or antigen 85B, previously shown to cross-react with HYB76-8. This capture ELISA permitted the identification of ESAT-6 expression from vaccinia virus constructs containing the esat-6 gene; this expression could not be identified by standard immunoblotting.     

Analysis of human antibody epitopes on the 65-kilodalton protein of Mycobacterium leprae by using synthetic peptides.

In order to study antibody reactivity to the Mycobacterium leprae 65-kilodalton (kDa) antigen, peptides representing overlapping sequences of the 65-kDa protein were synthesized, and a recombinant protein expression system for r65-kDa was constructed. Mouse monoclonal antibodies and leprosy patient seroreactivity to peptides and r65-kDa were tested by an enzyme-linked immunosorbent assay. All seven of the monoclonal antibodies used in this study reacted with their previously defined epitopes when tested against peptides. All monoclonal antibodies also reacted with r65-kDa. Leprosy patient seroreactivity to peptides and r65-kDa was seen in about one-third of active multibacillary cases. Specimens from patients positive for antibodies to peptides were seen to recognize different epitopes than did mouse monoclonal antibodies used in this study. It is concluded that substantial differences exist between mouse monoclonal antibodies and human leprosy patient reactivity to the 65-kDa antigen and that human seroreactivity to the 65-kDa antigen is indicative of a highly elevated bacillary load.     

Epitope analysis of the allergen ovalbumin (Gal d II) with monoclonal antibodies and patients' IgE.

Ovalbumin (OVA) is a major allergen (Gal d II) of hen egg white and is often the cause of hypersensitivity reactions to food. Further knowledge of the antigenic and allergenic epitopes of allergens will provide better treatment of this disease. To analyse these epitopes we produced a panel of monoclonal antibodies (mAbs) against native OVA. The initial information about the epitopes was obtained with the binding patterns of these mAbs in IEF-immunoprints and western blots of OVA under reducing and non-reducing conditions. It was possible to demonstrate that the different conformations of OVA exhibit different epitopes, and that there are other epitopes which are shared by each conformation. Seven different, although sometimes overlapping epitopes, could be determined on native OVA; four different epitopes on denaturated non-reduced OVA by means of immunoblots of the intact molecule. The number of epitopes which could be differentiated by the mAbs was increased by the use of peptide blots after CNBr fragmentation of the molecule. IgE binding to different OVA conformations and to CNBr-fragments of OVA was also detectable and appears in the same regions as the reactivity of some mAbs. Western blots of OVA and CNBr-peptides demonstrate that some antigenic/allergenic binding sites seem at least partly to be continuous epitopes. The identification of the CNBr-fragments was performed by a microsequence analysis of blotted CNBr-fragments after a 2-dimensional electrophoresis. IgE was found to bind the two largest CNBr-fragments (residues 41-172 and 301-385), but not the fragment corresponding to residues 173-196. A number of monoclonal antibodies also reacted with the two large fragments, especially with fragment 301-385, and some bind also to shorter peptides, such as fragment 173-196, which were not reactive to patients' IgE. Most of the monoclonal antibodies and patients' IgE bind to the fragments 41-172 and 301-385 in 2D-PAGE blots suggesting that these fragments are involved in an immunogenic structure.     

Monoclonal antibodies to an HIV-1 group O envelope recombinant

Monoclonal antibodies were developed to a recombinant HIV-I group O envelope protein derived from the isolate HAM112. These monoclonal antibodies were characterized for reactivity to a series of overlapping synthetic peptides (29-30 mers) covering gp120 C-terminal and gp41 ectodomain regions of the HIV-1 group O envelope protein. Most of these monoclonal antibodies reacted with peptides spanning sequences analogous to HIV-1 group M epitopes identified from studies in mice and humans. However, several of the antibodies that were nonreactive to individual peptides did react to a mixture of longer peptides from the N-terminal and C-terminal helical regions of the gp41 ectodomain. The monoclonal antibodies described in this study are valuable tools for characterization of antigenic differences between HIV-1 group O and group M viruses.     

Characterization of monoclonal antibodies to the globular domain of collagen IV.

Monoclonal antibodies were produced against NC1, the globular noncollagenous domain of collagen IV, isolated from bovine glomerular basement membrane. Cells from eight positive wells were cloned and the resulting monoclonal antibodies were studied in detail by immunofluorescence on human kidney sections, by Western blot and by ELISA against denatured subunits from NC1 hexamers and against native NC1 hexamers from different tissues. The monoclonal antibodies could be divided into two groups. Firstly, those monoclonal antibodies that, in ELISA and Western blot, reacted with peptides related to the alpha 1 chain of collagen IV and stained all basement membranes in the kidney. Secondly, a monoclonal antibody that, in ELISA and Western blot, reacted with peptides related to the Goodpasture antigen, the alpha 3 chain of collagen IV. When this antibody was applied to human kidney sections it stained the glomerular basement membrane very intensively. Bowman's capsule and some tubular basement membrane were also stained, although to a lesser extent. This staining pattern is the same as that observed with sera from patients with Goodpasture's syndrome. An attempt was made to separate different subtypes of the NC1 hexamer. A monoclonal antibody from the first group was used to make an affinity chromatography column. Glomerular basement membrane digested with collagenase was separated on this column and the collected fractions were analyzed by ELISA and SDS-PAGE. The result from this study support the idea that glomerular basement membrane is composed of at least two different subtypes of type IV collagen.     

MICA is a target for complement-dependent cytotoxicity with mouse monoclonal antibodies and human alloantibodies

The highly polymorphic major histocompatibility class I related chain A (MICA) gene encodes glycoproteins that have been shown to be expressed in epithelial cells, endothelial cells, keratinocytes, monocytes, and tumor cells. In previous experiments, we have studied MICA antigens using rabbit sera obtained by immunization with MICA peptides. We also found that several transplant recipients had specific antibodies against MICA in an ELISA assay with recombinant of MICA (r-MICA). In the present work we produced monoclonal antibodies by immunization of mice with recombinant MICA*008. Based on the different patterns of reactivity observed in ELISA, Western blot, and flow cytometry, mAbs 1.9C2, 2.4F5, 1.7AD, and 2.3D4 only reacted with denatured MICA and mAb 1.7A8 and 3.2H3 reacted also with native MICA as illustrated by flow cytometry with live cells. These monoclonal antibodies were postulated to bind to different sites of the MICA molecule. In order to investigate whether MICA expressed on the cell surface is able to mediate cell killing, antibody absorption, flow cytometry and complement-dependent cytotoxicity (CDC) were performed. We found that mouse monoclonal antibody 3.2H3 was able to kill 70% of HeLa cells. Absorption of a patient serum with pooled human platelets to remove antibodies against class I HLA resulted in a small shift of fluorescence and reduced killing from 100% to 70-75%. Absorption with the platelets and r-MICA produced a remarkable reduction in fluorescence staining and virtually reduced complement-dependent killing to the level of the negative controls. The results suggested that MICA alloantigens may be more immunogenic than could have been previously suspected.     

Production of monoclonal antibodies for detection of antigens in both their native and denatured states

Different immunization procedures were tested to find methods to enhance the proportion of monoclonal antibodies reacting equally well with native and denatured proteins for use in food analysis. Antibodies to soybean trypsin inhibitor with the desired characteristics were obtained by immunization with the denatured protein. Antibodies to ovomucoid were obtained by long‐term immunization. The monoclonal antibodies to ovomucoid were of the IgM class.     

Production and characterization of a peptide specific, anti-major histocompatibility complex class II, monoclonal antibody.

Monoclonal antibodies to major histocompatibility complex Class II proteins have been useful probes in understanding both the biochemistry and biology of these proteins. Almost all of the monoclonal antibodies previously described have been produced by immunization of mice with living cells. These antibodies react with native Class II proteins, but not usually with denatured material. It has been difficult to obtain specific anti-Class II antibodies which react with denatured proteins. Antibodies reactive with denatured proteins and with well-defined specificities would be useful in studies of Class II assembly and trafficking during the process of antigen presentation. In order to produce such an antibody we have immunized hamsters with a synthetic peptide corresponding to residues 146-177 (beta 1 domain) of the mouse A beta b protein. An antibody has been produced which reacts with the mouse Class II A beta chain from H-2b, H-2d, H-2p, and H-2q mice in immunoblotting assays, but not with the beta chain from H-2f, H-2j, H-2k or H-2s mice. Comparison of the amino acid sequences of these proteins along with the reactivity patterns of the antibody on synthetic peptides corresponding to homologous regions from A beta b, A beta k, A alpha b and Dp suggest that the region of 153 to 155 is critical for the reactivity of this antibody. This antibody does not react with native Class II protein found on the surface of living mouse cells.     

Production of mouse monoclonal antibodies that detect distinct neoantigenic epitopes on bound C3b and iC3b but not on the corresponding soluble fragments

Polyclonal antibodies raised in rabbits against sodium dodecyl sulphate (SDS)-denatured and reduced human complement factor C3 have in recent studies been shown to lack any reactivity towards native C3 but to react with antigens distinctly expressed by SDS-denatured C3 (C3(D) antigens). These antigens are also neoantigens specific for physiologically bound C3 and appear to be involved in the interaction of C3 with other complement components. The present investigation deals with production of mouse monoclonal antibodies against C3(D) antigens. To accomplish this two different immunization and screening procedures employing C3 preparations of known C3(D) expression were tested. From each group 14 clones were randomly selected and the reactivity of these and of a control group of 14 additional monoclonal anti-human C3 antibody preparations raised against native soluble C3 and C3b, was investigated in ELISA and immunoblotting. The procedure which employed denatured reduced C3 as both immunogen as well as screening antigen was shown to be superior for obtaining anti-C3(D) antibodies. Altogether 16 clones producing antibodies against C3(D) antigens were found. All of them bound to the C3 alpha-chain, 14 to C3c and one to C3d, and eight monoclonal antibodies specific for neoantigens of C3(D) type on bound C3b and/or iC3b were obtained. The majority of these detected neoantigenic epitopes in the 25,000 N-terminal fragment of the C3 alpha-chain specifically exposed by bound iC3b, but one monoclonal antibody was specific for the 36,000 C-terminal alpha-chain fragment and for both bound C3b and iC3b.     

Identification of a sequential B-cell epitope on major allergen (Cry j 1) of Japanese cedar (Cryptomeria japonica) pollen in mice

BACKGROUND: Japanese cedar (Cryptomeria japonica; CJ) pollinosis is one of the most common allergic diseases in Japan. B cell epitopes on Cry j 1, a major allergen of CJ pollen, have been analyzed by the specific monoclonal antibodies to Cry j 1, and most of these epitopes may be conformational, but no previous report has addressed the analysis of sequential epitope mapping with synthetic peptides. The main purpose of the present study is to identify IgE and IgG B cell epitopes on Cry j 1 by using a synthetic peptide approach in mice. METHODS: We synthesized 35 overlapping peptides that cover the entire length of Cry j 1 and examined whether mouse IgE and IgG antibodies produced by immunization with Cry j 1 reacted to the Cry j 1 peptides. RESULTS AND CONCLUSION: We found that mouse IgE and IgG antibodies reacted strongly to Cry j 1 peptide No. 15 ((141)GVEPVHPQDGDALTLRTATN(160)), though those antibodies did not react with other peptides. IgE and IgG antibody binding to peptide No. 15 was completely inhibited by Cry j 1 and the peptide. To determine the minimum epitope in peptide No. 15, we conducted an ELISA inhibition test. IgE and IgG antibody binding to peptide No. 15 was inhibited by smaller peptides of this peptide. We found the core of the epitope to be (145)VHPQDGDA(152).     

DNA immunization for the production of monoclonal antibodies to Blo t 11, a paramyosin homolog from Blomia tropicalis

BACKGROUND: Blo t 11 is a high molecular weight allergen from Blomia tropicalis with significant immunoglobulin (Ig)E binding frequency. Native and recombinant Blo t 11 are susceptible to degradation and the isolation and expression of the allergen is problematic thus obtaining sufficient amounts of purified Blo t 11 for antibody production is limiting. DNA-based immunization is an attractive alternative strategy that bypasses antigen purification for antibody production. OBJECTIVES: To use a DNA-based immunization protocol for the production and characterization of Blo t 11 monoclonal antibodies (mAbs). METHODS: The 2625 bp cDNA coding for Blo t 11 was cloned into a mammalian expression vector and immunized intramuscularly with electroporation into mice. Monoclonal antibodies to Blo t 11 were generated using a methylcellulose-based hybridoma cloning kit. These mAbs were utilized for native Blo t 11 isolation and the development of sandwich enzyme-linked immunosorbent assay (ELISA). RESULTS: Six mAbs recognizing the native and recombinant Blo t 11 were generated and characterized. Native Blo t 11 was affinity purified from Bt extract and its identity was confirmed by matrix assisted laser desorption/ionization - time of flight mass spectrometry. The native Blo t 11 showed IgE reactivity with 67% of mite allergic sera. A two-site ELISA developed showed a detection limit of 100 pg/ml of Blo t 11. CONCLUSION: A DNA-based immunization protocol was successfully used to generate Blo t 11 mAbs with a spectrum of distinct epitopes located throughout the whole molecule, and they are useful for immunoaffinity purification and immunoassays.     

Identification of surface-exposed linear B-cell epitopes of the nonfimbrial adhesin CS31A of Escherichia coli by using overlapping peptides and antipeptide antibodies.

As a first step toward the design of an epitope vaccine, by using the nonfimbrial adhesin CS31A of Escherichia coli as a carrier, a low-resolution topological and epitope map of the CS31A subunit was developed by using solid-phase peptide synthesis and polyclonal rabbit antibodies raised against both native and denatured proteins. Peptides constituting antigenic epitopes on the major subunit (ClpG) of the multimeric CS31A antigen were identified by examining the binding of the antibodies to 249 overlapping nonapeptides covering the amino acid sequence of ClpG. With antibodies raised against denatured ClpG subunit, seven major epitope regions, corresponding to residues 10 to 18, 45 to 58, 88 to 107, 148 to 172, 187 to 196, 212 to 219, and 235 to 241, were located. Most of the epitopes were hydrophilic and were located in variable regions, residing largely in loop regions at the boundaries of secondary structural elements of ClpG. In contrast, antibodies raised against native CS31A antigen reacted only with the peptide AVNPNA (positions 179 to 184), demonstrating that this peptide was the only linear B-cell epitope of the native protein. The different immunogenic profiles of native CS31A antigen and denatured ClpG indicated that the denaturation process resulted in marked conformational changes in the protein, which could expose epitopes hidden or absent in native CS31A. To identify the surface-exposed epitopes, nine peptides covering the dominant antigenic regions of ClpG were synthesized and used to prepare site-specific antibodies. Antipeptide antibodies were tested, in a competitive enzyme-linked immunosorbent assay (ELISA), for cross-reactivity with native CS31A and denatured ClpG subunit. Four of these antipeptide antibodies bound to the native protein in an accessibility ELISA, indicating that residues 44 to 56, 174 to 190, 185 to 199, and 235 to 249 were surface exposed on CS31A. These data indicate that an immunodominant surface-exposed linear epitope was present in the region from positions 179 to 184 of ClpG in the native CS31A antigen on intact bacterial cells and suggest that the four surface-exposed epitopes constitute potential sites for insertions or substitutions with heterologous peptides.     

Studies on the specificity of antibodies to ovalbumin in normal human serum: technical considerations in the use of ELISA methods.

As part of a study designed to reveal information about molecular features of allergenic food proteins after absorption from the gut the specificity of antibodies in normal human serum to hen's egg ovalbumin was investigated using ELISA techniques. Preliminary investigations with monoclonal antibodies and hyperimmune rabbit antiserum specific for ovalbumin in its native and denatured form established that the molecule underwent an extensive conformational change on adsorption to polyvinyl chloride microtitre plates. The native conformation could be retained by using antibodies to couple the protein to the surface. Serum from 90% of healthy adult human donors contained IgG antibodies to ovalbumin. In nearly all cases the antibodies were specific predominantly for the native molecule and could not be absorbed with denatured ovalbumin or peptides prepared from it by cleavage with cyanogen bromide or trypsin. Antibodies to denatured ovalbumin were detected in most sera but at very low levels and were preferentially absorbed by the homologous antigen; peptides and native ovalbumin showing variable absorptive activity. Thus, although ovalbumin is ingested largely in a denatured form, the serum antibody response is stimulated mainly by topographic epitopes of the native molecule.     

Immunochemical detection of Nepsilon-(carboxyethyl)lysine using a specific antibody

Methylglyoxal (MG) is generated through the Embden-Meyerhof and polyol pathways, and it rapidly reacts with proteins to form advanced glycation end products (AGE) such as N(epsilon)-(carboxyethyl)lysine (CEL). In the present study, polyclonal and monoclonal antibodies specific for CEL were prepared to estimate CEL content in aldehydes-modified proteins and the pathological localization in human kidneys. Polyclonal CEL-specific antibody was prepared by removing cross-reactive antibodies against N(epsilon)-(carboxymethyl) lysine (CML), one of the major AGE structures, using CML-conjugated affinity chromatography. Monoclonal CEL-specific antibody (CEL-SP) was obtained by immunization with CEL-bovine serum albumin, followed by successive screening according to CEL-RNase-positive but CML-RNase-negative criteria. A non-competitive ELISA showed that both the polyclonal and monoclonal CEL-specific antibodies significantly reacted with CEL-proteins but not with CML-proteins. A competitive ELISA also demonstrated that CEL-SP does not show cross-reactivity against CEL analogues such as CML, carboxymethylarginine (CMA) and S-carboxymethylcysteine (CMC), thus indicating that antibody is able to recognize the difference of one methyl group between carboxymethyl group and carboxyethyl group. Furthermore, CEL-SP significantly reacted with human serum albumin modified with MG but not with glyoxal or 3-deoxyglucosone, and its reactivity was highly correlated with the CEL content, which was determined by high performance liquid chromatography. Immunohistochemical studies using CEL-SP provided evidence that CEL-modified proteins accumulate in distal tubular epithelial cells of the diabetic rat. These results demonstrate that a specific antibody against CEL can be a powerful tool for detecting CEL both in vitro and in vivo.     

Specificity of monoclonal antibodies reactive with Fusobacterium nucleatum: effect of formalin fixation

Monoclonal antibody specific for Fusobacterium nucleatum was reacted with untreated and formalin fixed F. nucleatum cells by an enzyme-linked immunosorbent assay (ELISA) and by indirect immunofluorescence. Treatment of bacterial cells with formalin destroyed the antigenic determinant responsible for reactivity with this monoclonal antibody in both assays. Formalin fixation had no effect on hemagglutination activity (HA) of F. nucleatum cells or reactivity with polyvalent rabbit antiserum in double diffusion in agar. Scanning electron microscopy demonstrated that formalin fixation did not affect binding of F. nucleatum cells to microtiter plates. When developing monoclonal antibodies to be used as diagnostic reagents, the antigenic form utilized for immunization should be identical to the antigenic form which will eventually be used in the diagnostic assay.     

Topographic analysis of tobacco etch virus capsid protein epitopes

Monoclonal antibodies have been prepared, which react with capsid protein of an aphid-transmitted isolate of tobacco etch virus (TEV). Ten different monoclonal antibodies were characterized with reference to (1) antibody class, (2) reactivity with different plant virus antigens, (3) the spatial relationship between epitopes, and (4) whether these epitopes were located on the exterior surface of the virion. Three monoclonal antibodies were specific for TEV isolates. These monoclonal antibodies reacted with epitopes exposed on the external surface of the TEV particle. Seven monoclonal antibodies reacted with a variety of different potyviruses including TEV, potato virus Y, tobacco vein mottling virus, pepper mottle virus, watermelon mosaic virus II, and maize dwarf mosaic virus. In general, these seven monoclonal antibodies defined epitopes not readily accessible on the virion surface.