Characterization of human monoclonal antibodies selected with a hypervariable loop-deleted recombinant HIV-1(IIIB) gp120

Recombinant gp120 of the HIV-1(IIIB) isolate (BH10 clone) has been mutated to form the PR12 protein with the first 74 C-terminal amino acids and the V1, V2 and V3 hypervariable loops deleted. A variety of studies have shown that the CD4 binding domain (CD4bd) is very well exposed in PR12 in contrast to rgp120(LAI). Using PR12 for selection of human monoclonal antibodies (MAbs) from HIV-infected individuals, five MAbs were generated with specificities to the epitopes overlapping the CD4bd (1570A,1570C,1570D,1595 and 1599). The three MAbs, 1570A, C and D, generated from one HIV-infected individual, represent one MAb as determined by sequence analysis of the V(H)3 region. Since the epitopes overlapping the CD4bd exhibit variability among HIV-1 clades, the specificity of anti-CD4bd MAbs were distinguished by differing patterns of binding to recombinant envelope proteins derived from clade A, B, C, D and E viruses. The PR12-selected MAbs were also compared with a panel of gp120-selected anti-CD4bd MAbs and showed a different range of specificities. MAb 1599 is clade B specific, MAb 1595 reacts with the A, B and D clades, while MAb 1570 recognises the most conserved epitope, as it binds to all proteins. The results show that the exposure of different epitopes in the CD4bd of the PR12 protein allows this protein to serve as an immunogen and to induce anti-CD4bd antibodies.     

Epitope mapping of mouse monoclonal antibodies to the ppUL83 lower matrix phosphoprotein of human cytomegalovirus

Of nine mouse monoclonal antibodies (MAbs) directed against the lower matrix protein (pp65; ppUL83) of human cytomegalovirus (HCMV), all immunoprecipitated the 65-kDa protein. Only five were reactive by Western blotting, however, and four of these mapped to linear antigenic epitopes located between amino acids 184-195 (MAb C6), 343-357 (MAb C11), 448-462 (MAb C5), and 448-459 (MAb C13). The epitope specificity of the fifth antibody (MAb C3) and the four that recognised nonlinear sites could not be determined. Competition binding studies using HCMV antigen extracted from productively infected human embryonic lung fibroblasts (HELF), in an enzyme immunoassay (EIA), showed that three of the antibodies reactive with linear epitopes and two of those reactive with conformational epitopes (MAbs C3, C6, C11, C14, and C18), were unique in their binding specificities. MAb C4 competed with MAb C8 and MAb C5 competed with MAb C13 for binding to ppUL83. One of the linear epitopes identified, corresponding to amino acids SAFVFPTKDVAL (MAb C6), was an epitope described previously for CD8+ cytotoxic T lymphocytes.     

Characterization of monoclonal antibodies against the Escherichia coli hemolysin.

Twelve monoclonal antibodies (MAbs) produced against the Escherichia coli hemolysin (HlyA) encoded by the hemolysin recombinant plasmid pWAM04 were studied. HlyA derivatives from recombinant strains with different plasmids encoding HlyA amino-terminal and carboxy-terminal truncates, HlyA in-frame deletions, and HlyA frameshift mutations were used in immunoblots to localize the antigenic determinants for the anti-HlyA MAbs. The mapping of the MAb epitopes was also facilitated by immunoblotting analysis of HlyA polypeptide fragments derived by cyanogen bromide cleavage. The HlyA epitopes for 11 of the MAbs were mapped to relatively small linear regions of the cytolysin ranging from 28 to 160 amino acids. Five of the MAbs (C10, G8, E2, B7, and D12) neutralized HlyA hemolytic activity to varying degrees. The epitopes for these neutralizing MAbs were found to reside within the following HlyA regions: C10 and G8, amino acids 2 to 160; E2, amino acids 161 to 194; B7, amino acids 518 to 598; and D12, amino acids 626 to 726. Hemolytically active HlyA was dependent on the action of the hlyC gene product. The D12 MAb recognized only HlyA produced by strains with an intact hlyC function. MAb A10 recognized an epitope within the HlyA region from amino acids 728 to 829 where a glycine-rich repeat domain exists; however, this MAb did not neutralize HlyA hemolytic activity. A HlyA domain map showing the anti-HlyA epitope location was constructed.     

Characterization of three novel monoclonal antibodies against hepatitis C virus core protein

Three novel monoclonal antibodies (MAbs) were established against a recombinant hepatitis C virus (HCV) core protein derived from cloned genotype 1b HCV cDNA. MAbs C7-50 and C8-59 recognize a conserved linear epitope represented by amino acid residues 21 to 40 of the nucleocapsid protein. MAb C8-48 is directed against a strain-specific conformational epitope located within the first 82 amino acids. A sensitive two-site MAb-based immunoradiometric assay was established using antibodies directed against distinct epitopes on the nucleocapsid protein. Processed 21 kDa core protein was detected by immunoblotting in human hepatocellular carcinoma cell lines and primary adult rat hepatocytes transfected with a cytomegalovirus promoter-driven expression construct. Immunofluorescence microscopy studies revealed a granular and vesicular cytoplasmic staining pattern. MAb C7-50 was used successfully to detect HCV core antigen in chronically infected chimpanzee liver tissue. These MAbs represent important reagents for the study of HCV biology and for the development of immunodiagnostic assays. © 1996 Wiley-Liss, Inc.     

Rat monoclonal antibodies to nonoverlapping epitopes of human immunodeficiency virus type 1 gp120 block CD4 binding in vitro.

Monoclonal antibodies (MAbs) to a recombinant form of the envelope glycoprotein gp120 of human immunodeficiency virus type 1 (HIV-1 IIIB) were raised in rats and screened for their ability to block recombinant gp120 binding to recombinant, soluble CD4 (sCD4) in vitro. Four such MAbs were identified and characterised. Each MAb bound strongly to gp120 from eight widely divergent HIV-1 strains from the United States and Africa. Two MAbs were mapped to the fourth conserved (C4) region of gp120, whereas the other two recognised an as yet undefined, conformationally sensitive epitope. MAbs to the latter epitope were the more potent in blocking the gp120-sCD4 interaction. None of the MAbs, however, had potent neutralising activity.     

Epitope mapping of monoclonal antibodies capable of neutralizing cytotoxic necrotizing factor type 1 of uropathogenic Escherichia coli

Cytotoxic necrotizing factor type 1 (CNF1) of uropathogenic Escherichia coli belongs to a family of bacterial toxins that target the small GTP-binding Rho proteins that regulate the actin cytoskeleton. Members of this toxin family typically inactivate Rho; however, CNF1 and the highly related CNF2 activate Rho by deamidation. Other investigators have reported that the first 190 amino acids of CNF1 constitute the cellular binding domain and that the CNF1 enzymatic domain lies within a 300-amino-acid stretch in the C terminus of the toxin. Amino acids 53 to 75 appear to be critical for cell receptor recognition, while amino acids Cys866 and His881 are considered essential for deamidation activity. To delineate further the functional domains of CNF1, we generated 16 monoclonal antibodies (MAbs) against the toxin and used them for epitope mapping studies. Based on Western blot immunoreactivity patterns obtained from a series of truncated CNF1 proteins, this panel of MAbs mapped to epitopes located throughout the toxin, including the binding and enzymatic domains. All MAbs showed reactivity to CNF1 by Western and dot blot analyses. However, only 7 of the 16 MAbs exhibited cross-reactivity with CNF2. Furthermore, only three MAbs demonstrated the capacity to neutralize toxin in either HEp-2 cell assays (inhibition of multinucleation) or 5637 bladder cell assays (inhibition of cytotoxicity). Since CNF1 epitopes recognized by neutralizing MAbs are likely to represent domains or regions necessary for the biological activities of the toxin, the epitopes recognized by these three MAbs, designated JC4 (immunoglobulin G2a [IgG2a]), BF8 (IgA), and NG8 (IgG2a), were more precisely defined. MAbs JC4 and BF8 reacted with epitopes that were common to CNF1 and CNF2 and located within the putative CNF1 binding domain. MAb JC4 recognized an epitope spanning amino acids 169 to 191, whereas MAb BF8 mapped to an epitope between amino acids 135 and 164. Despite the capacity of both MAbs to recognize CNF2 in Western blot analyses, only MAb BF8 neutralized CNF2. MAb NG8 showed reactivity to a CNF1-specific epitope located between amino acids 683 and 730, a region that includes a very small portion of the putative enzymatic domain. Taken together, these findings identify three new regions of the toxin that appear to be critical for the biological activity of CNF1.     

Location and architecture of an antibody-binding site of influenza A virus nucleoprotein

Amino acid positions recognized by monoclonal antibodies (MAbs) in the influenza A virus nucleoprotein (NP) have been reported. As these residues were scattered in the three-dimensional (3D) structure of NP, no patterns of the architecture of antibody-binding sites could be inferred. Here, we used site-specific mutagenesis and ELISA to screen the amino acids surrounding position 470 recognized by the MAb 3/1 as a linear epitope. Ten amino acid residues involved in the reaction of NP with the MAb 3/1 and the MAb 469/6 were identified. Our data are the first to outline a compact site recognized by MAbs in the 3D structure of the influenza virus NP.     

Monoclonal antibodies directed against AFAP-110 recognize species-specific and conserved epitopes.

The actin filament-associated protein, AFAP-110, is a Src SH2/SH3 binding partner that can modulate changes in actin filament structure. AFAP-110 contains a carboxy terminal motif that facilitates actin filament interactions, as well as amino terminal protein binding motifs, including an SH3 binding motif, two SH2 binding motifs, and two Pleckstrin homology domains. Two monoclonal antibodies (MAbs) were developed that recognized epitopes in either the amino terminus (MAb 4C3) or the carboxy terminus (anti-AFAP-110) of AFAP-110. Site-directed mutations that change key proline residues to alanine in the SH3 binding motif and an adjacent proline-rich motif abrogated MAb 4C3 binding. These same mutations have been shown to prevent SH3 interactions between AFAP-110 and Src527F. These data indicate that MAb 4C3 recognizes an epitope that is part of the SH3 binding motif. Interestingly, MAb 4C3 is not efficiently reactive with mammalian homologs of AFAP-110. Sequence analysis of a putative cDNA clone that encodes the amino terminus of the human AFAP-110 isoform predicted a one amino acid difference within this epitope, indicating a mechanism for species-specific binding by MAb 4C3. A second, MAb anti-AFAP-110, recognizes AFAP-110 across species and binds to an epitope within the carboxy terminus. This epitope includes the 5th heptad repeat of the carboxy terminal, leucine zipper motif (amino acids 592-598)--a motif that facilitates self-associations and may regulate the function of AFAP-110. These MAbs will be useful for analyzing the effects of AFAP-110 upon cell morphology and actin filament integrity. In addition, the avian-specific MAb 4C3 may be useful for studying the effects of avian AFAP-110 constructs expressed in mammalian cells, by providing an internal epitope tag.     

Monoclonal antibodies against NIH 3T3 cells transformed by human thyroid carcinoma DNA

First cycle transformants of NIH 3T3 cells transfected with metastatic human thyroid carcinoma DNA were used as immunogen to obtain monoclonal antibodies (MAbs) against normal and transformation-related antigens. The transformed cell line (M33) was shown to contain Alu sequences. Two MAbs were selected on the basis of their differential reactivity toward untreated NIH 3T3 cells or the transformed M33 cell line. By immunofluorescence, immunoelectronmicroscopy and biochemical analysis, the first MAb (MTr1) was demonstrated to recognize an epitope on cytoskeletal filaments of proliferating murine fibroblasts. Similar MTr1-labelled filaments were also found to accumulate into cytoplast-like structures spontaneously produced by M33 cells. The characterization by immunofluorescence of MTr2, the second MAb, indicates that it recognizes a specific human antigen associated with normal thyroid epithelial cells and differentiated thyroid tumors.     

Resistance of mice vaccinated with rabies virus internal structural proteins to lethal infection

Summary Mice were vaccinated with recombinant vaccinia virus (rVac) expressing the glycoprotein (G), nucleoprotein (N), phosphoprotein (NS) or matrix protein (M) of rabies virus and their resistance to peripheral lethal infection with street rabies virus was examined. Mice vaccinated with rVac-G or rVac-N developed strong antibody responses to the corresponding proteins and essentially all mice survived challenge infection. Mice vaccinated with rVac-NS or rVac-M developed only a slight antibody response, however, a significant protection (59%) was observed in the rVac-NS-vaccinated mice, whereas rVac-M-vaccinated mice were not protected. No anti-G antibodies were detected in the sera of mice which had been vaccinated with rVac-N or rVac-NS and survived challenge infection. Passive transfer of anti-N monoclonal antibodies (MAbs) recognizing an epitope located on amino acids 1–224 of the protein prior to challenge resulted in significant protection, although the protection was not complete even with a high amount of antibodies. In contrast, none of the mice given MAbs recognizing an epitope of amino acids 247–415 or F(ab)₂ fragments from a protective MAb IgG were protected. Administration of anti-CD 8 MAb to rVac-N-vaccinated mice showed no significant effect on protection. Our observations suggest that a considerable part of the protection achieved by the vaccination with rVac-N can be ascribed to the intact anti-N antibodies recognizing an epitope located on amino acids 1–224 of the protein.     

Phenotypes and protein-epitope phenotypic variation among fresh isolates of Trichomonas vaginalis.

Fresh isolates of Trichomonas vaginalis were examined for reactions to a panel of five monoclonal antibodies (MAbs). Four MAbs (C20A3, DM126, DM116, and C55) were to distinct surface immunogens and one MAb (L64) was to a cytoplasmic component. The fresh isolates were evaluated by indirect immunofluorescence (IF), immunoblotting, and radioimmunoprecipitation. IF assay with C20A3 MAb gave isolates which were homogeneous nonstaining (negative [Neg] phenotype) and isolates which were heterogeneous staining and nonstaining (positive [Pos] and Neg phenotype, respectively) organisms. Immunoblotting and radioimmunoprecipitation assays revealed that surface phenotypic heterogeneity among isolates with C20A3 MAb was due to the presence or absence of the immunogen from the parasite surface. IF assay with DM126 MAb also gave Pos and Neg phenotypes among parasites of some isolates. All of the isolates were always Neg phenotype with DM116 and C55 MAbs. The occurrence of Neg phenotype organisms with DM126, DM116, and C55 was due to epitope inaccessibility to their respective MAbs and not to the absence of the immunogen from trichomonal membranes. All isolates possessed the cytoplasmic protein recognized by L64 MAb. Paired isolates (taken 5 to 6 days apart) from 24 women were also studied. Four of the 24 paired isolates (16%) had different phenotype distributions at the two timepoints for C20A3. Fresh isolates also underwent phenotypic variation during in vitro growth and multiplication, as determined with C20A3. Also, 7 of the 24 paired isolates demonstrated dramatic changes in the accessibility of DM126 MAb to epitope binding. Lastly, 55 (90%) of 60 serum samples from patients with trichomoniasis evaluated in this study possessed antibody to the C20A3 reactive molecule. The data show that the fresh T. vaginalis isolates were predominantly Neg phenotype and confirm the occurrence of protein and epitope phenotypic variation for major immunogens among fresh isolates of the pathogenic human trichomonads.     

Monoclonal antibodies against human basic fibroblast growth factor

Recombinant human basic fibroblast growth factor (hbFGF) was used as an antigen to develop, by a somatic cell fusion technique, four monoclonal antibodies (MAbs), that recognize the complete and amino-terminal truncated form of hbFGF. Isotype identification showed that MAbs designated MAb12 and MAb98 were IgG1; and those designated MAb52 and MAb78 were IgG2b. All these MAbs bound the complete form of hbFGF produced in E.coli. Competition with synthetic polypeptides, a replication of 1-9 aa and of 141-146 aa of hbFGF, and truncated forms of hbFGF by 13 and 40 amino acid residues in its amino-terminal produced in E. coli by recombinant technique, revealed at least two epitopes recognized by the four IgG type MAbs. MAb12 and MAb78 recognized the epitope located within the first 9 amino acid residues at the amino terminal of the complete hbFGF. MAb52 and MAb98 recognized the one located between the amino acid residue no. 14 and 40. None of MAbs bound bovine acidic FGF (aFGF). Using MAb52 or MAb98 and MAb78, a two-site EIA has been developed. This EIA is sensitive enough to detect 0.5 ng/ml of hbFGF. Furthermore, MAb78 was used as a ligand for affinity chromatography to purify hbFGF mutein CS4, which binds weakly to a heparin affinity column.     

A novel monoclonal antibody against FbaA can inhibit the binding of the complement regulatory protein factor H to group A streptococcus

Some microbial pathogens utilize human complement regulatory proteins, such as factor H (FH) and factor H-like protein 1 (FHL-1), for immune evasion. FbaA is an FHL-1 and FH binding protein expressed on the surface of group A streptococcus (GAS), a common agent of pharyngeal, skin, and soft tissue infections. In this study, we prepared monoclonal antibodies (MAbs) against FbaA, assayed them for specificity, and located their binding domains in FbaA. We found an MAb called FbaA MAb2, which demonstrated the highest affinity to GAS among all of the MAbs. Based on the binding with component peptides, the detected epitope, which was specific for FbaA MAb2, was the amino acid residues 95 to 118 of FbaA; on the other hand, it did not bind with the truncated protein of the internally deleted residues of the segment from 95 to 118 of FbaA. Furthermore, the predominant amino acids specific for FbaA MAb2 screened by phage display epitope library were I, T, P, D, and L, corresponding to the amino acid residues 101, 103, 105, 106, and 110 of FbaA, respectively. The binding location of FbaA with FH and FHL-1 was a 16-amino-acid region corresponding to amino acid residues 97 to 112 of FbaA, which overlapped the FbaA MAb2 binding domain, as confirmed by competitive inhibition enzyme-linked immunosorbent assay and immunofluorescence microscopy. Based on the results of the invasion assay, FbaA MAb2 can inhibit the binding of FH to GAS.     

Protection of immunosuppressed mice against infection with Pseudomonas aeruginosa by recombinant P. aeruginosa lipoprotein I and lipoprotein I-specific monoclonal antibodies.

Outer membrane protein I (OprI) is one of the major proteins of the outer membrane of Pseudomonas aeruginosa. The protective effect of OprI vaccination and that of three OprI-specific monoclonal antibodies (MAbs) against infection with P. aeruginosa were tested in immunosuppressed mice. The combination of Oprl and MAb 2A1 protected the mice against a challenge with a 96-fold 50% lethal dose. The binding site of MAb 2A1 was mapped, resulting in the identification of a protective epitope (amino acids 7 to 20).     

Two simple and rapid methods to detect monoclonal antibodies with identical epitope specificities in a large population of monoclonal antibodies.

Two novel and simple methods have been developed for detecting monoclonal antibodies (MAbs) with identical epitope specificities from a large population of MAbs against human chorionic gonadotropin (hCG). The first method was based on the observation that following radioiodination many molecules of an antigen are altered in such a manner that they cannot be recognized by MAbs. The proportion of radiolabelled antigen (Bmax) able to bind to a MAb was characteristic of that MAb and MAbs having identical specificities showed identical Bmax values. Using this principle it was possible to identify MAbs having identical epitope specificities within a large population of MAbs against hCG. In the second method one test MAb was immobilized on a plastic surface through an immunochemical bridge. This MAb was then incubated with 125I-hCG previously complexed with a second MAb. Such a complex could bind to the solid phase MAb only if the two MAbs were not identical. The results obtained with both methods were concordant. With such methods it is possible to identify MAbs with identical epitope specificities immediately after the initial screening of the fusion. These methods do not require subcloning, ascites production, or the purification and iodination of large number of MAbs.     

Analysis of Linear B-Cell Epitopes of the Nucleoprotein of Ebola Virus That Distinguish Ebola Virus Subtypes

Ebola virus consists of four genetically distinguishable subtypes. We developed monoclonal antibodies (MAbs) to the nucleoprotein (NP) of Ebola virus Zaire subtype and analyzed their cross-reactivities to the Reston and Sudan subtypes. We further determined the epitopes recognized by these MAbs. Three MAbs reacted with the three major subtypes and recognized a fragment containing 110 amino acids (aa) at the C-terminal extremity. They did not show specific reactivities to any 10-aa short peptides in Pepscan analyses, suggesting that these MAbs recognize conformational epitope(s) located within this region. Six MAbs recognized a fragment corresponding to aa 361 to 461 of the NP. Five of these six MAbs showed specific reactivities in Pepscan analyses, and the epitopes were identified in two regions, aa 424 to 430 and aa 451 to 455. Three MAbs that recognized the former epitope region cross-reacted with all three subtypes, and one that recognized the same epitope region was Zaire specific. One MAb, which recognized the latter epitope region, was reactive with Zaire and Sudan subtypes but not with the Reston subtype. These results suggest that Ebola virus NP has at least two linear epitope regions and that the recognition of the epitope by MAbs can vary even within the same epitope region. These MAbs showing different subtype specificities might be useful reagents for developing an immunological system to identify Ebola virus subtypes.     

Analysis of linear B-cell epitopes of the nucleoprotein of ebola virus that distinguish ebola virus subtypes

Ebola virus consists of four genetically distinguishable subtypes. We developed monoclonal antibodies (MAbs) to the nucleoprotein (NP) of Ebola virus Zaire subtype and analyzed their cross-reactivities to the Reston and Sudan subtypes. We further determined the epitopes recognized by these MAbs. Three MAbs reacted with the three major subtypes and recognized a fragment containing 110 amino acids (aa) at the C-terminal extremity. They did not show specific reactivities to any 10-aa short peptides in Pepscan analyses, suggesting that these MAbs recognize conformational epitope(s) located within this region. Six MAbs recognized a fragment corresponding to aa 361 to 461 of the NP. Five of these six MAbs showed specific reactivities in Pepscan analyses, and the epitopes were identified in two regions, aa 424 to 430 and aa 451 to 455. Three MAbs that recognized the former epitope region cross-reacted with all three subtypes, and one that recognized the same epitope region was Zaire specific. One MAb, which recognized the latter epitope region, was reactive with Zaire and Sudan subtypes but not with the Reston subtype. These results suggest that Ebola virus NP has at least two linear epitope regions and that the recognition of the epitope by MAbs can vary even within the same epitope region. These MAbs showing different subtype specificities might be useful reagents for developing an immunological system to identify Ebola virus subtypes.     

The use of anti-IgG monoclonal antibodies in mapping the monocyte receptor site on IgG

Monoclonal antibodies (MAbs) directed against epitopes on the Cγ1, Cγ2, Cγ3 and Cγ2-Cγ3 interface regions of human IgG were used to attempt to localize the monocyte Fc receptor (FcR) binding site. The MAbs have been assayed for their capacity to inhibit the interaction between ¹²⁵I-labelled IgG (¹²⁵I-IgG) and human monocytes or human histiocytic lymphoma U937 cells. Two MAbs specific for epitopes on the N-terminal region of the Cγ2 domain, and one MAb recognizing an epitope in the Cγ2-Cγ3 inter-domain region inhibited binding of ¹²⁵I-IgG to monocyte FcRs. The remaining MAbs, against a C-terminal Cγ3 domain epitope, another Cγ2/Cγ3 region epitope and the Glm(f) allotope on the Cγl domain did not inhibit the interaction. The capacity of the MAbs to bind to their respective epitopes on cell surface FcR-bound IgG was also studied, using indirect radiobinding and immunofluorescence assays. All of the MAbs, except those with Cγ2 domain specificities, were able to detect FcR-bound IgG under these conditions. The results confirm the role of the Cγ2 domain in the interaction of IgG with monocytes and demonstrate that epitopes in the Cγ3 and Cγ2-Cγ3 regions are not involved in the binding site.     

Effect of cross-reactivity of alpha-fetoprotein monoclonal antibody on quantitation of serum AFP and radioimmunodetection of hepatocellular carcinoma.

Murine hybridomas were generated by immunizing mice with purified alpha-fetoprotein (AFP) to determine the cross-reactivity of AFP antibodies with human serum albumin (HSA). Eighteen out of 23 hybridoma products were cross-reactive with HSA. All 23 monoclonal antibodies (MAbs) could be subgrouped into 3 groups by their binding affinity to AFP and HSA, and one MAb from each group, IIB3, IIIB1 and IIIC6, were chosen for this study. The affinity to AFP was highest for IIB3 followed by IIIC6 and IIIB1, and that to HSA was in the order IIIB1 greater than IIB3. Immunohistochemical staining revealed no cross-reactions of these antibodies with HSA at the tissue level. IIB3 was the most efficient in the quantitative assays, indicating high affinity and specificity (non-cross-reactivity) of the antibodies, both of which are prerequisites for in vitro assays. Scans with good tumor localization were obtained from mice which received 131I-labeled IIB3 or IIIC6 after xenografting of the human hepatocellular carcinoma. The images obtained with IIB3 were inhibited in the presence of either AFP or HSA in the circulation. These findings indicated that the use of cross-reactive and/or high-affinity antibodies against AFP provides an inhibitory factor for tumor localization. MAb IIIC6, because of its high specificity and moderate affinity to AFP, seems to be the best candidate for immunoscintigraphy of the tumors in future studies.     

Antigenic Importance of the Carboxy-Terminal Beta-Strand of the Porcine Reproductive and Respiratory Syndrome Virus Nucleocapsid Protein

Five domains of antigenic importance were previously mapped on the nucleocapsid protein (N) of the porcine reproductive and respiratory syndrome virus (PRRSV), and a domain comprised of the 11 C-terminal-most amino acids (residues 112 to 123) was shown to be essential for binding of N-specific conformation-dependent monoclonal antibodies (MAbs). In the present study, the importance of individual residues within this C-terminal domain for antigenicity was investigated using eight different mutant constructs of N expressed in HeLa cells. Single amino acid substitutions were introduced into the C-terminal domain of the N protein, and the significance of individual amino acids for MAb reactivity was determined by immunoprecipitation. None of the MAbs tested recognized the mutant with a leucine-to-proline substitution at residue 114 (L114P), while V112P, R113P, R113D, I115P, and R116P reduced MAb binding significantly. Conversely, substitution of amino acids at positions 118 (T118S) and 121 (P121A) had little effect on MAb binding. Secondary-structure predictions indicate that amino acids 111 to 117 form a beta-strand. In view of the fact that replacement of beta-strand-forming amino acids with proline elicited the greatest effect on MAb binding, it appears that secondary structure in the C terminus of the N protein is an important determinant of conformational epitope formation. While the crystal structure of the PRRSV N protein remains to be determined, results from these studies broaden our understanding of the secondary structures that make up the PRRSV N protein and shed some light on how they may relate to function.