Characterization of epitopes recognized by monoclonal antibodies to aphid transmissible and non-transmissible strains of turnip mosaic virus

Summary Fourteen monoclonal antibodies (MAbs) were prepared against two strains of turnip mosaic virus (TuMV) differing in aphid transmissibility. Serological specificity of fourteen MAbs against the two strains was tested by indirect ELISA. Three MAbs were able to distinguish aphid transmissible TuMV strain 1 from non-aphid transmissible strain 31 while four MAbs reacted only with strain 31. No cross-reactivity between the two strains was found using these specific MAbs. Based upon the ability of Mab to inhibit the reaction of other MAbs, antibody competition test indicated that fourteen MAbs recognized six different epitopes on the virus particle; MAbs specific to strain 1 recognized two epitopes while MAbs specific to strain 31 also recognized two epitopes. The remaining two epitopes are common. Since the six amino acid differences between the coat proteins of the two strains were found at the N-terminal regions, MAbs specific to strain 1 or 31 bound to the different epitopes on the N-terminal regions in coat proteins of the two strains.     

Immunochemical studies of tobacco mosaic virus—VII. Use of comparative surface accessibility of residues in antigenically related viruses for delineating epitopes recognized by monoclonal antibodies

The binding properties of 18 monoclonal antibodies (MAbs) directed against the tobacco mosaic virus (TMV) coat protein were studied with five related tobamoviruses and seven mutant viruses, as well as with the dissociated coat proteins of these variants. Ten of the antibodies bound to both TMV and TMV protein, but these were able to discriminate between different mutants only when whole virus particles were compared in the immunoassay. Three MAbs reacted with TMV but not with dissociated viral subunits and these recognized the same residue exchanges. Five MAbs recognized synthetic peptides of 13–28 residues corresponding to parts of the protein. By comparing the common accessible surface between TMV and antigenically related viruses, it was possible to narrow down the region recognized by some of these MAbs. The linear peptide sequence recognized by these antibodies did not represent the entire epitope and residue exchanges around this region were able to affect the binding of MAbs.     

Partial antigenic characterization of different potato virus Y NTN strain isolates

Eight isolates of potato virus Y NTN strain (PVY-NTN) of different origin were studied by means of monoclonal antibodies (MAbs) in non-competitive and competitive enzyme-linked immunosorbent assay (ELISA), and by immunoblot analysis of the viral coat protein (CP). As the MAbs reacted with the denatured viral CP, their epitopes must be continuous. The ELISA data demonstrate that the epitopes are topologically different. The epitopes may be located on the N-terminal part of CP as showed its partial amino acid sequencing and the immunoblot analysis.     

Nucleotide sequences of the helper component-proteinase genes of aphid transmissible and non-transmissible isolates of turnip mosaic virus

Summary We have compared nucleotide sequences of the helper component-proteinase (HC-Pro) coding region of aphid transmissible (isolate 1) and non-transmissible (isolate 31) isolates of turnip mosaic virus (TuMV). HC-Pro coding regions of both TuMV isolates 1 and 31 were 1,374 nucleotide long. The nucleotide sequence homology between these isolates was 93.5%, with 89 nucleotides substitution. The nucleotides of HC-Pro regions of two isolates of TuMV genomes encoded 458 amino acids of M_r 51,746 (isolate 1) and M_r 51,764 (isolate 31). The deduced amino acid sequence homology between these isolates was 98.7% with six different amino acids. These amino acids appeared to regulate the activity of HC-Pro needed for aphid transmissibility of TuMV.     

Epitope mapping of Grapevine virus A capsid protein

Summary.  Previously characterized monoclonal antibodies (MAbs) to Grapevine virus A (GVA) showed a differential reactivity against intact or partially destabilized virus particles [2]. In the present study, this differential reactivity was confirmed and several peptides reacting with a panel of four different antibodies were identified by the PEPSCAN method of epitope mapping. Oligopeptide sequences comprised between coat protein residues 61 (V) and 72 (T) were recognized by all the antibodies tested. One of these peptides (VGPKASK) was also reactive when expressed on recombinant phage particles as a fusion protein with protein pVIII. The specificity of this sequence for antibody binding was also demonstrated by competitive-ELISA using one of the GVA MAbs. The results of this study suggest that GVA particles carry a highly structured epitope centered on a common peptide region of the coat protein sequence. Received June 5, 2001 Accepted October 26, 2001     

Strain analysis and epitope mapping of West Nile virus using monoclonal antibodies.

Monoclonal antibodies (MAbs) against an Indian strain (804994) and an Egyptian strain (E 101) of West Nile virus (WNV) were prepared in mice. Nine MAbs against the 804994 strain and 5 MAbs against E 101 strain were obtained. All 14 MAbs reacted with the envelope (E) protein of WNV in an immunoblot assay. They were tested by an enzyme-linked immunosorbent assay (ELISA) for their cross-reactivity with WNV, Japanese encephalitis virus (JEV) and Dengue-2 virus (DEN-2), and for their reactivity in haemagglutination-inhibition (HAI) test. Based on these results MAbs were broadly grouped into three groups, namely WNV-specific HAI-positive, WNV-JEV cross-reactive HAI-positive, and WNV-JEV cross-reactive HAI-negative MAbs. The antigenic cross-reactivity between twelve WNV strains isolated from different geographical regions and their respective hosts was assessed using these MAbs in HAI and complement fixation (CF) tests. The strain analysis by CF distinguished Indian from South African strains. However, a similarity between some Indian and South African strains in HAI was observed. E 101 strain appeared to have antigenic similarity with Indian as well as South African strains. Overall it appears that antigenically similar strains of WNV are prevalent in India. A single heterogenous domain was apparent on the epitope map of WNV deduced by ELISA additivity test.     

Three epitopes located on the coat protein amino terminus of viruses in the bean common mosaic potyvirus subgroup

Summary.  Twenty-seven of 29 strains of viruses in the bean common mosaic virus (BCMV) subgroup of legume-infecting potyviruses reacted strongly with one or more of the monoclonal antibodies (MAbs) which are known to be specific for epitopes located along the 50 amino acids which constitute the N-terminal end of the viral coat protein. Approximately one half of the virus strains reacted with the N-terminal epitope specific (NTES) MAb 4G12 which is specific for epitope E/B4, while the other half reacted with NTES MAbs 4 A1 or 4F9 which are specific for epitope E/B3. All but two strains contained at least one of these epitopes while no strain contained both. Competitive assays using five sequential, non-overlapping, synthetic, 10mer peptides indicated that the amino acids critical for epitope E/B3 reaction were located at positions 5, 7, and 10 from the N-terminal end of the coat protein. By deduction we postulate that the amino acids critical for epitope E/B4 are located at positions 10, 16, and 17. Because epitope E/B3 requires isoleucine at position 10 for expression whereas epitope E/B4 requires valine to be expressed, no one strain can express both epitopes. Two viruses in our tests (azuki mosaic and Dendrobium mosaic viruses) had deletions in this portion of their sequence explaining their failure to react MAbs specific for either epitope. The critical amino acids for a third epitope, E/B3A, were located at positions 16 and 17. We found no correlation between any of the three N-terminal epitopes defined in this study and the presence or absence of any biological property that we could accurately measure: i.e., symptomatology, host range, or pathotype. However, when coat protein sequences were aligned according to epitope type E/B3 or E/B4, we found that sequences within groups had high levels of identity while between group identities were low. We also found that sequences in the 3′-end non-coding region exhibited similar relationships within and between epitope groups. Two strains of BCMV (NL-4 and RU-1) were found to possess coat protein sequences typical of epitope E/B4 but 3′-NCR sequences typical of epitope E/B3. These data suggest that both strains may be the result of natural recombinants between the two epitope groups. Received May 18, 1998 Accepted October 26, 1998     

Immunochemical studies of tobacco mosaic virus--V. Localization of four epitopes in the protein subunit by inhibition tests with synthetic peptides and cleavage peptides from three strains

Two antigenic determinants have been located in the vicinity of residues 1-10 and 34-39 of the coat protein of tobacco mosaic virus by means of ELISA and complement fixation inhibition experiments with synthetic peptides. The determinant present in residues 1-10 is also expressed at the outer surface of the subunits when they are assembled into virions. Two other determinants expressed in the dissociated protein subunit have been located in the vicinity of residues 55-61 and 80-90 by means of inhibition tests with tryptic peptides. These results bring to seven the number of antigenic determinants of the protein subunit that have been located.     

Common Peptide Epitope in Mumps Virus Nucleocapsid Protein and MHC Class II-Associated Invariant Chain

The present study describes a 7 amino acid-long sequence (YQQQGRL) which is identical in HLA-associated invariant chain and mumps virus nucleocapsid protein and is additionally followed by one conservative amino acid pair. As such a long amino acid homology is extremely rare in two evolutionarily unrelated proteins the possibility that it could induce immunological cross-reactivity was evaluated. Several antigenicity indices suggested high antigenic potential within this region. Synthetic peptides containing this sequence were reactive with 31% of monoclonal antibodies specific for mumps virus nucleocapsid protein in ELISA. High antibody levels against this epitope were found in 7% of mumps-seropositive human sera and antibody levels clearly increased after natural mumps infections and mumps vaccinations. Rabbit antibodies raised against a synthetic invariant chain peptide AYF-LYQQQGRLDKL-C reacted with corresponding nucleocapsid peptide RFAKYQQQGRLEAR-C and antibodies against the nucleocapsid peptide reacted with the invariant chain peptide. Rabbit antibodies against the invariant chain peptide also reacted with nucleocapsid molecules in formaldehyde-fixed mumps virus-infected cells, and antibodies against the nucleocapsid peptide reacted with invarianl chains expressed in methanol-fixed cells. One monoclonal antibody specific for the nucleocapsid molecule also reacted with cells expressing invariant chains. In immunoprecipitation rabbit antibodies against the invariant chain peptide bound to invariant chains while antibodies against the nucleocapsid peptide did not. The results suggest tha t there is antigenic similarity in mumps virus nucleocapsid molecule and HLA-associated invariant chain which may cause immunological cross-reactivity between these molecules.     

Use of Luminex xMAP-derived Bio-Plex bead-based suspension array for specific detection of PPV W and characterization of epitopes on the coat protein of the virus

A panel of monoclonal antibodies (MAbs) directed against the N-terminus region of the coat protein (CP) of strain PPV W (isolate 3174) was generated by immunizing mice with recombinant peptides. The best performing MAbs were identified as 2C3 and 10G7. MAb 2C3 was selected for comparison of a standard TAS-ELISA protocol with a Luminex xMAP technology-derived bead-based suspension array system described as a triple antibody sandwich-microsphere immunoassay (TAS-MIA). TAS-MIA was as sensitive as TAS-ELISA for the specific detection of PPV W in herbaceous and woody hosts. It was completed in 4h, and used less reagents. Epitope recognition analysis was carried out using a set of overlapping synthetic pin-bound peptides (Mimotopes). Peptides (2)DEEDD(6) and (46)MFNPV(50) were the epitopes recognized most commonly by the best performing MAbs. Linear epitope prediction of B-cell recognition sites confirmed that both peptides fall within highly antigenic and accessible regions. The second glutamic acid residue of the epitope is crucial for MAb recognition, and the context of the epitope is as important as the sequence of the epitope. The results obtained in ELISA, Western blot, and TAS-MIA correlated with B-cell recognition prediction. This is an effective approach to identify suitable antigenic epitopes that generate antibodies for use in reliable diagnostic procedures. This is the first report of the detection of a plant virus using the Luminex xMAP bead-based suspension array system.     

Identification of an epitope on the dengue virus membrane (M) protein defined by cross-protective monoclonal antibodies: design of an improved epitope sequence based on common determinants present in both envelope (E and M) proteins

Summary.  The protective capacity of monoclonal antibodies (MAbs) generated to the dengue-2 virus envelope (E) and premembrane (prM) proteins was tested in vivo. Two anti-E MAbs, 2C5.1 and 4G2 and two anti-prM MAbs, 2A4.1 and 2H2 provided cross-protection against all four dengue virus serotypes. Overlapping sets of synthetic peptides spanning amino-acid sequence 301–401 (domain III) of the E protein and the entire prM protein were then used to locate their epitopes. The anti-E MAbs strongly reacted with the peptide sequence 349-GRLITVNPIVT-359 (E349–359) from domain III and the immunodominant epitope, 274-SGNLLFTGHL-283 (E274–283) from the hinge region between domains I and II. The anti-prM MAbs strongly reacted with the sequence, 40-PGFTVMAAIL-49 (M40–49) from the first membrane-spanning domain of the M protein. These anti-prM MAbs also reacted with peptides E274–283 and E349–359, while the anti-E MAbs reacted with a peptide sequence, 1-FHLTTRNGEP-10 from the prM protein and these cross-reactions with both proteins were confirmed using immunoblot assays. MAbs 2C5.1, 4G2 and 2H2 more strongly reacted with an MEH1 peptide GLFTPNLITI, which was designed as an antigenic hybrid between these E and prM peptide sequences, than with any of these natural peptide sequences. These peptide sequence will now be tested for their ability to generate cross-protective antibodies against each dengue virus serotype when delivered with appropriate T-helper epitopes. Accepted August 13, 1999     

Antigenic properties of phage displayed peptides comprising disulfide-bonded loop of the immunodominant region of HIV-1 gp41

The HIV-1 envelope glycoprotein gp41 contains Cys(X)5Cys motif, which has been shown to elicit a strong antibody response in almost all HIV-1 infected individuals. This disulfide-bonded loop region is conserved in most retroviruses suggesting the existence of an essential function in virus life cycle. In this study, we displayed the peptides comprising 12 amino acids of the immunodominant loop of gp41 on the surface of M13 phage as N-terminal fusions to the minor coat protein pIII and major coat protein pVIII of the phage and demonstrated that cysteine loop containing peptide expressed on phage recognized 62 out of 63 (98.4%) HIV-1 positive samples but not control negative sera while phage bearing linear peptides detected 4-30% of HIV-1-positive sera. The main advantage of phage-based ELISA or other antibody detection-based diagnostic tests of HIV-infection to be used for massive screening in developing countries is the reproducible, simple, rapid and low-cost production of recombinant antigens.     

Characterization and application of a series of monoclonal antibodies against SARS-CoV-2 nucleocapsid protein

The ongoing coronavirus disease 2019 (COVID-19) pandemic has a significant global social and economic impact, and the emergence of new and more destructive mutant strains highlights the need for accurate virus detection. Here, 90 monoclonal antibodies (MAbs) that exclusively reacted with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (NP) were generated. These MAbs did not cross-react with NPs of common human coronaviruses (HCoVs, i.e., 229E, OC43, HKU1, and NL63) and Middle East Respiratory Syndrome Coronavirus. Subsequently, overlapped peptides in individual fragments (N1–N4) of NP were synthesized. N1-3 (25-GSNQNGERSGARSKQ-39), N3-1 (217-AALALLLLDRLNQL-230), and N4-8 (393-TLLPAADLDDFSKQL-407) were identified as major epitopes using enzyme-linked immunoassay (ELISA) and recognized by 47, 1, and 18 MAbs, respectively. The 24 remaining MAbs exhibited no reactivity with all synthetic peptides. Among MAb-epitope pairs, only MAbs targeting epitope N1-3 displayed no cross-reaction with NPs of SARS-CoV-1 and other SARS-related CoVs. All Omicron variants contained a three-amino acid deletion (31ERS33) in the N1-3 region. Thus, MAbs targeting N1-3 failed to recognize these variants. Furthermore, a double-antibody sandwich ELISA for antigen detection was established using the optimal MAbs. Overall, a series of MAbs targeting SARS-CoV-2 NP was prepared, characterized with epitope mapping, and applied for the detection of SARS-CoV-2 antigens, and some novel B-cell epitopes of the viral NP were identified.     

Antipeptide monoclonal antibodies inhibit the binding of rabies virus glycoprotein and alpha-bungarotoxin to the nicotinic acetylcholine receptor

It has been reported that binding to muscle nicotinic acetylcholine receptor at the post-synaptic membrane is an important event of the rabies virus neurotropism. The binding site can be located within the 190-203 region of the virus glycoprotein sharing a high degree of homology with the "toxic loop" of the curare-mimetic snake neurotoxins. We have synthesized a tetradecapeptide corresponding to this glycoprotein region and used it, following conjugation with an immunogenic carrier to raise MAbs. We found that some MAbs raised against the peptide were able to recognize both the virus glycoprotein and the snake neurotoxin alpha-bungarotoxin; moreover, they can inhibit the binding of rabies virus glycoprotein and alpha-bungarotoxin to the nicotinic acetylcholine receptor extracted from the electric organs of Torpedo marmorata. On the basis of this cross-reactivity, we suggest that rabies virus glycoprotein and curare-mimetic snake neurotoxins share three-dimensionally similar structures in order to bind to the nicotinic cholinergic receptor. The potential use of the immunogenic properties of the peptide for the rational design of a synthetic vaccine against rabies is proposed.     

Immunochemical studies of turnip yellow mosaic virus--II. Localization of a viral epitope in the N-terminal residues of the coat protein

The N-terminal region of the coat protein of turnip yellow mosaic virus has been shown by immunochemical means to be present on the external surface of the virion. Seven synthetic peptides corresponding to overlapping regions of residues 1–12 of the coat protein have been prepared. The inhibitory activity of these peptides was measured by complement fixation tests, RIA and ELISA, using either antibodies directed to the depolymerized viral subunits or antibodies to the capsid. The activity of peptides varied markedly in different assays, illustrating the advantage of using several different immunochemical methods for the analysis of viral antigenic determinants.     

Characterisation and epitope analysis of monoclonal antibodies to virions of clover yellow vein and Johnsongrass mosaic potyviruses

Summary Mouse monoclonal antibodies (MAbs) against the Australian B strain of clover yellow vein (C1YVV-B) and the JG strain of Johnsongrass mosaic (JGMV) potyviruses were produced, characterised and the epitopes with which they reacted were deduced. Using intact particles of C1YVV a total of ten MAbs were obtained which reacted strongly with C1YVV-B in an enzyme-linked immunosorbent assay and Western blots. Four of these MAbs (1, 2, 4, and 13) were found to be ClYVV-specific, as they reacted with all five C1YVV strains from Australia and the U.S.A. but not with 11 strains of bean yellow mosaic (BYMV), pea mosaic (PMV), and white lupin mosaic (WLMV) viruses which, together with C1YVV, form the BYMV subgroup of potyviruses. These MAbs failed to react with eight other potyvirus species, including six which infect legumes like the viruses in the BYMV subgroup. The C1YVV MAb 10 was found to be BYMV subgroup-specific. It reacted strongly with 15 of the 16 strains of viruses in the subgroup and gave no reaction with eight other potyviruses. The other five C1YVV MAbs reacted with varying degrees of specificity with the BYMV subgroup viruses and also with other potyviruses. Eight of the C1YVV MAbs (1, 2, 4, 5, 13, 17, 21, and 22) reacted with the intact coat proteins only and not with the truncated (minus amino terminus) coat protein of C1YVV suggesting that the epitopes for these MAbs are located in the surface-exposed, amino-terminal region of the C1YVV coat protein. Comparison of published coat protein sequences of BYMV and C1YVV isolates indicated that the epitopes for the four ClYVV-specific MAbs may be in the amino-terminal region spanning amino acid residues 18 to 30, whereas those for the other four MAbs may be located in the first 17 amino-terminal amino acid residue region. The epitopes that reacted with BYMV subgroup-specific MAb 10 and MAb 30 which reacted with 20 of the 24 potyvirus isolates, are probably located in the core region of C1YVV coat protein as these MAbs reacted with the intact as well as truncated coat protein of C1YVV. Analysis, in Western blot immunoassay, of 17 MAbs raised against virions of JGMV revealed that only two MAbs (1–25 and 4–30) were JGMV-specific, whereas others displayed varying degrees of specificity to different potyviruses. When these MAbs were screened against the intact and truncated (minus 67 amino-terminal amino acid residues) coat proteins of JGMV, the two JGMV-specific MAbs reacted only with the intact coat protein, whereas the other MAbs reacted with the intact as well as with truncated coat proteins, in Western blots. These results suggest that the epitopes for the two JGMV-specific MAbs are located in the surface-exposed amino-terminal 67 amino acid residue region and those for the cross-reactive MAbs are contained in the conserved core region of the JGMV coat protein. Screening of potyvirus MAbs against intact and truncated coat proteins thus appears to be a simple procedure to select virus-specific MAbs to potyviruses.     

Antibodies elicited by a biosynthetic peptide related to a major immunogenic area of FMDV A12.

Foot-and-mouth disease virus (FMDV) capsid contains 60 copies each of four structural proteins, virus proteins 1-4. Virus protein 1 (VP1) plays an important immunogenic role, being the only VP that is immunogenic as an isolated protein. Even peptides representing a partial amino acid (AA) sequence of VP1 can induce protective immunity in experimental hosts. A 32 AA residue, in a tandem repeat configuration (32dimer), of sero/subtype A-12 Lp ab VP1 (AA 132-168) was highly immunogenic for its homologous subtype and partially protective for FMDV serotype A strain A24. This cross-reactivity was further demonstrable in the ELISA and mouse protection tests. Three different antibody populations were isolated by affinity chromatography (AFC) from the serum of a steer immunized with the 32dimer. Each population seems to recognize a different epitope on the 32dimer peptide since each fraction was defined as unique by its reactivity with different subtypes of FMDV virus in RIA, ELISA, neutralization and competition assays. Considering the neutralizing activity of each of the antibody populations the pattern of neutralization of the 32dimer elicited antiserum can be described. Two of the three epitopes were mapped by competition assays using synthetic peptides.     

MicroELISA method for the determination of thymosin beta 9 discriminating between thymosin beta 9 and the structurally closely related thymosin beta 4

In order to obtain specific antibodies against thymosin beta 9 showing minimal cross-reactivity with the highly homologous peptide thymosin beta 4, the N-terminal fragment 1-14 of thymosin beta 9 was used for immunization. These antibodies have been tested in a competitive ELISA and show less than 1% cross-reactivity with thymosin beta 4. On the other hand, antibodies raised against the native thymosin beta 9 (1-14) cross-react 35% with thymosin beta 4. Specific antibodies against thymosin beta 9 are important for studying the concentration and localization of thymosin beta 9 in thymus and other bovine tissues because thymosin beta 9 is always accompanied by thymosin beta 4. Using N-terminal fragments of thymosin beta 4-like peptides may be a general approach for obtaining specific antibodies since this part of sequence is less conserved in thymosin beta 4-like peptides.     

Monoclonal antibodies against the major coat protein of filamentous phage as a useful analytical tool for bacteriophage peptide/protein display

Four mouse monoclonal antibodies (MAbs) that react with filamentous M13KO7 and R408 phage were obtained. Three of these MAbs (two IgG2a and one IgG3) recognize linear sequences of the p8 main structural coat protein, and one (IgG2a) identifies a putatively conformational epitope, as suggested by Western blot. These MAbs also react with recombinant phage expressing peptide antigens fused to p8, and are though useful reagents for peptide/protein phage display screening based methods. The latter was shown in an enzyme-linked immunoadsorbent assay (ELISA) and a visual immunoassay where one of the anti-p8 MAbs was used to capture recombinant phages displaying a peptide characteristic of the Hepatitis B virus surface antigen or a Dengue virus-related peptide antigen.     

Epitope characterization by modifications of antigens and by mapping on resin-bound peptides. Discriminating epitopes near the C-terminus and N-terminus of Escherichia coli ribosomal protein S13

The feasibility of using antigen modifications and synthetic resin-bound peptides to distinguish closely related epitopes has been demonstrated in this report. Epitopes recognized by five monoclonal antibodies (MAbs) specific to Escherichia coli ribosomal protein S13 have been located near the C-terminus and N-terminus of the S13 molecule; these epitopes were characterized by modifications of antigens and by utilization of peptides of increasing length synthesized on aminomethyl crosslinked polystyrene resin. Three of these MAbs react with the C-terminal peptide S13(84-117) which has five Lys residues clustered within the last 16 amino acids. Phthalylation of Lys residues almost eliminated the binding of two MAbs and reduced binding of the third by 50%. Removal of the C-terminal Lys residue(s) at the S13 C-terminus with carboxypeptidase B has no effect on the binding of these three MAbs. A 23-residue peptide corresponding to S13(95-117) was synthesized by a modified Merrifield solid phase method. Samples of resin with peptides of increasing length were obtained after each cycle of amino acid coupling. The peptides were deprotected without hydrolysis of the peptide-resin linkage and used in an enzyme immunoassay to detect the extent of MAb interaction with the lengthening peptides. The epitopes recognized by the two MAbs more sensitive to Lys modification have been localized in S13(97-117). The third MAb binds to S13(98-117) but binds more strongly when the sequence is lengthened. Two MAbs directed toward the N-terminal 22 residues of S13 were similarly characterized. Binding of one MAb, little affected by phthalylation, required the N-terminal residue of S13 to be present in the synthetic peptide. The other MAb, whose binding was inhibited by phthalylation, bound to the synthetic S13(2-22) and bound more strongly to the synthetic S13(1-22).