Analysis of peptidic epitopes recognized by the three monoclonal antibodies specific for the same region of glycophorin A but showing different properties.

Analysis of epitopes for the three monoclonal antibodies (GPA105, GPA33, OSK4-1) against glycophorin A (GPA) was performed with the use of proteolytic fragments of GPA, the synthetic nonapeptide with the sequence of amino acid residues 35-43 of GPA, and a series of peptides synthesized on plastic pins. The antibodies were specific for a short peptide sequence RAHE (a.a. 39-42 of GPA, MAbs GPA105 and OSK4-1) or RAHEV (a.a. 39-43 of GPA, MAb GPA33). Despite recognizing the same fragment of GPA, the three antibodies showed differences in fine specificity and in response to antigen desialylation. Reactions with single replacement analogs of the RAHEV sequence showed that immunodominant (unreplaceable) residues for the MAbs GPA33 and OSK4-1 were His and Glu, respectively, whereas no such residue was found for the MAb GPA105. Desialylation of the antigen gave strong enhancement of reactivity with the MAb GPA33, moderate--with the MAb GPA105, and weak or no enhancement of reaction with the MAb OSK4-1. The results showed that monoclonal antibodies directed against the same fragment of the polypeptide chain of densely glycosylated antigen may recognize different subsites which are masked at different degree by sialic acid residues.     

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.     

A族链球菌(GAS)Fba蛋白单克隆抗体对应表位核心氨基酸的确定

目的:对A族链球菌Fba蛋白McAb2所对应的表位进行定位.方法:以初步定位的表位区段为线索合成三段重叠多肽,dot-ELISA检测McAb2与合成肽的亲合力,并以McAb2为靶分子,利用噬菌体随机七肽库进行亲和筛选,用竞争ELISA鉴定阳性克隆.结果:dot-ELISA检测结果表明Fba_(100-112)肽段与McAb2的结合能力最强.经过3轮亲和筛选后,随机挑选20个噬菌体克隆,竞争ELISA对其与McAb2的亲和力做检测,其中12个克隆显示较强的阳性结果.阳性克隆DNA测序,与Fba基因第100位氨基酸至110位氨基酸中ITPDL同源性较高.结论:通过dot-ELISA将A族链球菌Fba蛋白McAb2对应的表位定位于100～112位氨基酸,结果同噬菌体随机肽库筛选的核心氨基酸位置吻合.为进一步研制表位肽疫苗、研究Fba蛋白及其单克隆抗体的生物学功能奠定了基础.     

Newly characterized species-specific immunogenic Chlamydophila pneumoniae peptide reactive with murine monoclonal and human serum antibodies

A monoclonal antibody (MAb) directed against an unknown Chlamydophila pneumoniae epitope has been characterized, and the respective peptide mimotope has been identified. A murine MAb specific for C. pneumoniae was used to select peptides from phage display libraries. The peptides identified from the phage display library clones reacted specifically with the respective target murine MAb and with human sera previously identified as having antibody titers to C. pneumoniae. The selected peptide mimotope sequences tended to be composed of charged residues surrounding a core of hydrophobic residues. The peptide with the best binding could inhibit >95% of binding to the MAb, suggesting that the selected peptide binds the paratope of the respective MAb. The peptide reacted with human sera previously determined by microimmunofluorescence to have anti-C. pneumoniae antibodies. The peptide was competitively competed with the MAb against Renografin-purified, sonicated C. pneumoniae in an enzyme-linked immunosorbent assay and with whole-cell C. pneumoniae in an indirect fluorescence assay format, demonstrating its potential utility in the development of diagnostics. The use of this novel peptide may allow investigators to establish standardized assays free from cross-reactive Chlamydia trachomatis and Chlamydophila psittaci epitopes and immunoreactivity.     

Identification of B-cell epitope of dengue virus type 1 and its application in diagnosis of patients

Using a serotype-specific monoclonal antibody (MAb) of dengue virus type 1 (DEN-1), 15F3-1, we identified the B-cell epitope of DEN-1 from a random peptide library displayed on phage. Fourteen immunopositive phage clones that bound specifically to MAb 15F3-1 were selected. These phage-borne peptides had a consensus motif of HxYaWb (a = S/T, b = K/H/R) that mimicked the sequence HKYSWK, which corresponded to amino acid residues 111 to 116 of the nonstructural protein 1 (NS1) of DEN-1. Among the four synthetic peptides corresponding to amino acid residues 110 to 117 of the NS1 of DEN-1, -2, -3, and -4, only one peptide, EHKYSWKS (P14M) of DEN-1, was found to bind to 15F3-1 specifically. Furthermore, P14M was shown to inhibit the binding of phage particles to 15F3-1 in a competitive inhibition assay. Histidine(111) (His(111)) was crucial to the binding of P14M to 15F3-1, since its binding activity dramatically reduced when it changed to leucine(111) (Leu(111)). This epitope-based peptide demonstrated its clinical diagnostic potential when it reacted with a high degree of specificity with serum samples obtained from both DEN-1-infected rabbits and patients. Based on these observations, our DEN-1 epitope-based serologic test could be useful in laboratory viral diagnosis and in understanding the pathogenesis of DEN-1.     

Immunological characterization of an immunomodulatory epitope in S-antigen/arrestin with a sequence motif common to tumor necrosis factor alpha.

Some monoclonal antibodies (mAbs) to retinal S-antigen recognize a phylogenetically conserved epitope (S2) in the N-terminal part of the protein. These antibodies have been shown to inhibit the induction of experimental autoimmune uveoretinitis by S-antigen in rats. Using Pepscan method, we localized this epitope on the amino acid (aa) residues 40-50, i.e., PVDGVVLVDPE (peptide S2). MAb binding was confirmed by ELISA, competition-ELISA and dot blot. Other S-antigen peptides with homologies to epitope S2 and peptides exhibiting the pathogenic and T-cell proliferation inducing sites did not bind these mAbs. Epitope S2 displays an immunological crossreactivity with human tumor necrosis factor (TNF) alpha. Recent results indicate that both peptide S2 and a peptide from human TNF alpha (aa residues 31-53) containing the common sequence motif GVxLxD induce TNF alpha production in monocytes. We analyzed the fine structure of the common epitope by studying mAb binding in an amino acid residue exchange experiment.     

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).     

Human granulocyte-macrophage colony-stimulating factor (hGM-CSF): identification of a binding site for a neutralizing antibody.

One approach to the localization of functionally active regions of human granulocyte-macrophage colony-stimulating factor (hGM-CSF) is to map the epitopes recognized by neutralizing anti-hGM-CSF monoclonal antibodies. We have defined the epitope recognized by one neutralizing antibody (LMM102) using proteolytic fragments obtained by enzymic digestion of bacterially synthesized hGM-CSF. RP-HPLC fractionation of a tryptic digest resulted in the identification of an immunoreactive "tryptic core" peptide containing 66 amino acids (52% of the protein). Further digestion of this "tryptic core" with S. aureus V8 protease produced a unique immunoreactive hGM-CSF product comprising two peptides, residues 86-93 and 112-127, linked by a disulfide bond between residues 88 and 121. The individual peptides, generated by reduction with dithiothreitol, were not recognized by the antibody. An analog of this peptide has been synthesized chemically and shown to have similar immunoreactivity to the epitope obtained by enzymic digestion. A series of modified peptides has also been synthesized to identify further the region required for antibody recognition.     

Fine manipulation of antibody affinity for synthetic epitopes by altering peptide structure: antibody binding to looped peptides*.

Linear peptides weakly imitate antibody binding sites on globular proteins when the peptides are shown to be effective at all. As a step toward enhancing the ability of peptides to mimic epitopes, we have examined the effects of various alterations in peptide structure on antibody binding. Synthetic peptides containing the core amino acid sequence of residues 41 to 48 from horse cytochrome c were examined for their ability to bind antibodies elicited against the 41-48 peptide coupled to bovine serum albumin (BSA). Since residues 41-48 in native cytochrome c are part of an omega loop, in some peptides cysteines were incorporated for intrachain disulfide bonding to stabilize loop structure. In additional cases, glycine was incorporated as a spacer between the natural sequence and the cysteine residues with the intent of relaxing loop structure slightly. Eleven analogues containing the 41-48 sequence were tested. These included native cytochrome c and the 1-80 and 1-65 cyanogen bromide-cleaved fragments. The native protein did not bind the anti-41-48 antibodies. The other analogues differed by over three orders of magnitude in their binding. The affinity of binding was inversely related to the extent of predicted loop structure indicating that the antibodies were elicited against the 41-48 sequence in a more unfolded conformation despite the Pro Gly sequence at positions 44 and 45 that generally favors a beta turn. Surprisingly, the immunizing peptide, containing residues 41-48 only, was the poorest binding peptide. The relative impotence of 41-48 was shown to be largely due to differences at the amino terminus between the free and BSA-coupled peptides as the antibodies were elicited against the latter. The distinctions among the synthetic peptides containing the 41-48 sequence show the exquisite sensitivity of antibody binding to amino acid changes that may occur outside of an epitope and suggest modifications in peptide structure at the periphery of an epitope that can lead to desired changes in antibody affinity.     

Identification of an antigenic epitope in Helicobacter pylori urease that induces neutralizing antibody production

We previously reported a mouse monoclonal antibody (MAb), termed L2, specific for Helicobacter pylori urease strongly inhibited its enzymatic activity. Here, to gain insight into how this antibody affects urease activity, the epitope that was recognized by the antibody was determined. By screening a panel of overlapping synthetic peptides covering the entire sequence of the two subunits (UreA and UreB), we identified a stretch of UreB-derived 19 amino acid (aa) residues (UB-33; aa 321 to 339, CHHLDKSIKEDVQFADSRI) that was specifically recognized by the L2 antibody. Further sequential amino acid deletion of the 19-mer peptide from either end allowed us to determine the minimal epitope as 8 amino acid residues (F8; SIKEDVQF) for L2 reactivity. This epitope appears to lie exactly on a short sequence which formed a flap over the active site of urease, suggesting that binding of the L2 antibody sterically inhibits access of urea, the substrate of urease. Finally, immunization of rabbits with either the 19-mer peptide or the 8-mer minimal epitope resulted in generation of antiurease antibodies that were capable of inhibiting the enzymatic activity. Since urease is critical for virulence of H. pylori, antigenic peptides that induce production of antibodies to inhibit its enzymatic activity may potentially be a useful tool as a vaccine for prevention and treatment of H. pylori infection.     

Effects of histidine residues on adsorption equilibrium of peptide antibodies.

Histidine and arginine residues in a peptide, which represent residues 112-121 of myoglobin, were replaced by alanine, and antibodies for these peptides were raised in rabbits. Adsorption equilibria between these peptides and antibodies were measured to identify the essential amino acid residues for specific binding and their effects on adsorption behavior. Protonation of one histidine residue affected strongly the dependence of adsorption equilibrium on pH, and the adsorption capacities of the antibodies against the peptides containing this essential histidine residue rapidly decreased at around pH 5-6, which corresponds to the pKa value of histidine. Immunization with peptide sequences containing such histidine residues is one method to obtain suitable antibodies for immunoaffinity chromatography, which have a weak affinity for the elution step under mild conditions.     

Immunochemical Characterization of the MPB70/80 and MPB83 Proteins of Mycobacterium bovis

MPB70 and MPB80 (MPB70/80) and MPB83 are closely related antigens which are highly expressed in Mycobacterium bovis. MPB70/80 are soluble secreted antigens, while MPB83 is an exported lipoprotein associated with the bacterial surface. In the present study, these antigens had different mobilities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions. These differences may be explained by the fact that MPB70 and MPB83 both have two internal cysteine residues which would create ring structures by disulfide bonding. We analyzed the structures of MPB70/80 and MPB83 by using monoclonal antibodies (MAbs) raised against bovine purified protein derivative or whole M. bovis cells. MAb 1-5C reacted specifically with MPB70 and MPB80, and MAb MBS43 reacted specifically with MPB83, while the other antibodies, including several previously described MAbs, bound all three antigens. MAbs and polyclonal antibodies reacted strongly with reduced protein and less well with nonreduced protein, indicating involvement of linear epitopes. Epitopes of MAbs Bov-1, 2-6B, 1-5C, and 1-1D were mapped by using synthetic peptides of MPB70. Sequence comparison showed the peptide with the 1-5C-reactive epitope to have three residues different from those in the homologous region of MPB83. Exchanges of A for S in position 112 or Q for E in position 116 abolished the reactivity of MAb 1-5C. Polyclonal rabbit antibodies to native purified MPB70 reacted strongly with peptides 6, 7, and 8 of the N-terminal half of mature MPB70. Cattle sera of experimentally M. bovis-infected animals recognized a broader spectrum of peptides. These findings indicate that there is diagnostic potential for these proteins and that there is also a possible role for antibodies in elucidation of the host-mycobacterium relationship involving a surface-bound and exposed lipoprotein, MPB83, and its highly homologous soluble secreted MPB70/80 counterparts.     

Localization of two epitopes recognized by monoclonal antibody PCG-4 on Clostridium difficile toxin A.

The toxin A gene of Clostridium difficile contains a 2.5-kb region encoding a series of contiguous repeating units located at the COOH terminus of the molecule. We previously showed that the monoclonal antibody (MAb) PCG-4, which neutralizes the enterotoxic activity of toxin A, binds to epitopes located within these repeating units. In the present study, we subcloned a series of fragments from this portion of the gene. The recombinant peptides expressed from the gene fragments were examined for reactivity with MAb PCG-4 to identify the epitopes involved in binding. Our results showed that MAb PCG-4 recognizes epitopes in amino acid residues 2097 through 2141 and amino acid residues 2355 through 2398.     

Localization of the virus neutralizing and hemagglutinin epitopes of E1 glycoprotein of rubella virus.

Current serological assays using whole rubella virus (RV) as a target antigen for detecting RV-specific antibodies fail to define specific RV proteins and antigenic determinants such as hemagglutinin (HA) and virus-neutralizing (VN) epitopes of rubella virus. A panel of E1 deletion mutants and a subset of E1-specific monoclonal antibodies (MAb) were used for the initial analysis of HA and VN epitopes of E1 glycoprotein. A peptide region (E1(193) to E1(269)) was found to contain HA and VN epitopes. Using both overlapping synthetic peptides and truncated fusion proteins within this region, the HA epitope defined by MAb 3D9F mapped to amino acid residues E1(214) to E1(240), while two VN epitopes defined by MAb 21B9H and MAb 16A10E mapped to amino acid residues E1(214) to E1(233) and E1(219) to E1(233), respectively. The epitopes defined in this study are recognized by antibody whether or not the epitopes are glycosylated.     

Characterisation of the epitope for a herpes simplex virus glycoprotein B-specific monoclonal antibody with high protective capacity

Monoclonal antibody (MAb) 2c, specific for glycoprotein B of herpes simplex virus (HSV), had been shown to mediate clearance of infection from the mucous membranes of mice, thereby completely inhibiting mucocutaneous inflammation and lethality, even in mice depleted of both CD4(+) and CD8(+) cells. Additionally, ganglionic infection was highly restricted. In vitro, MAb 2c exhibits a potent complement-independent neutralising activity against HSV type 1 and 2, completely inhibits the viral cell-to-cell spread as well as the syncytium formation induced by syncytial HSV strains (Eis-Hübinger et al. in Intervirology 32:351-360, 1991; Eis-Hübinger et al. in J Gen Virol 74:379-385, 1993). Here, we describe the mapping of the epitope for MAb 2c. The antibody was found to recognise a discontinuous epitope comprised of the HSV type 1 glycoprotein B residues 299 to 305 and one or more additional discontinuous regions that can be mimicked by the sequence FEDF. Identification of the epitope was confirmed by loss of antibody binding to mutated glycoprotein B with replacement of the epitopic key residues, expressed in COS-1 cells. Similarly, MAb 2c was not able to neutralise HSV mutants with altered key residues, and MAb 2c was ineffective in mice inoculated with such mutants. Interestingly, identification and fine-mapping of the discontinuous epitope was not achieved by binding studies with truncated glycoprotein B variants expressed in COS cells but by peptide scanning with synthetic overlapping peptides and peptide key motif analysis. Reactivity of MAb 2c was immensely increased towards a peptide composed of the glycoprotein B residues 299 to 305, a glycine linker, and a C-terminal FEDF motif. If it could be demonstrated that antibodies of the specificity and bioactivity of MAb 2c can be induced by the epitope or a peptide mimicking the epitope, strategies for active immunisation might be conceivable.     

Identification of Residues within the Herpes Simplex Virus Type 1 Origin-Binding Protein That Contribute to Sequence-Specific DNA Binding

Gene UL9 of herpes simplex virus type 1 encodes an 851-amino-acid protein which is essential for viral DNA synthesis and functions as a sequence-specific origin-binding protein and DNA helicase. We generated monoclonal antibodies against purified UL9 protein and identified one such antibody (MAb 13924) that can block the interaction of the UL9 C-terminal DNA-binding domain (amino acids 534–851) with its recognition sequence. MAb 13924 interacted with immobilized peptides containing residues 780–786 of UL9. Although the corresponding region of the homologous protein encoded by varicella-zoster virus differs at only a single position it was not recognized by MAb 13924. Site-directed mutagenesis experiments confirmed that residues within this region contribute to the epitope recognized by MAb 13924 and may be involved in sequence-specific DNA binding. In addition, all eight lysine residues within the DNA-binding domain were separately changed to alanine and the DNA-binding properties of the mutated proteins were examined. The results showed that lysine residues that are located close to the peptide recognized by MAb 13924 or lie within the region of the DNA-binding domain most highly conserved among homologous alphaherpesvirus proteins play a role in sequence-specific DNA binding. Moreover, alteration of a lysine residue 18 amino acids from the recognized peptide prevented the interaction of MAb 13924 with the UL9 C-terminal DNA-binding domain. Three helical segments are predicted to occur within the region containing mutations that affect sequence-specific binding and interaction with MAb 13924.     

Short synthetic peptides derived from viral proteins compete with HIV gp120 for the binding to CD4 receptors

In the complex mechanism of adhesion, internalization, and infection of cells by human immunodeficiency virus (HIV) viral particles, a determinant role is played by the viral envelope glycoprotein gp120, which binds to CD4 receptors of T cells and monocytes. We tested the ability of a panel of 7- to 12-residue synthetic peptides, selected from the region 414-434 of the HIV-1 gp120, to inhibit the binding of the viral protein to CD4 receptors of cultured human lymphoid cells. The assay was based on the observation that the binding of gp120 to the receptors interferes with the binding of a specific anti-CD4 monoclonal antibody, as a result of the masking of the antibody epitope; thus, we tested whether preincubation of cells with the peptides before gp120 addition might restore the recognition of the CD4 molecule by the antibody. High expression of CD4 receptors was thus assumed as indication that the binding of the viral protein had been inhibited. Maximum activity was displayed by a 9-residue peptide located near the amino terminal end of the 414-434 fragment. In addition, several fragments deduced from other viral proteins, possessing partial amino acid sequence homology with the HIV gp120 fragment, exhibited a similar type of interaction with the CD4 receptor. All active peptides contain the Cys residue (position 423 of gp120). This residue is essential, although not sufficient, for inhibiting gp120 binding, as few other amino acid residues within the fragment play a complementary role in increasing or decreasing the inhibitory ability.     

Analysis of the extracellular processing of HIV-1 gp160-derived peptides using monoclonal antibodies specific to H-2Dd molecule complexed with p18-I10 peptide

The immunodominant peptide of human immunodeficiency virus 1 gp 160 for murine cytotoxic T cells of H-2d haplotype, has been originally identified as a 15 amino acid residue peptide P18IIIB (RIQRGPGRAFVTIGK) (Takahashi et al., 1988). Further studies have indicated that a more active form of the peptide is generated by removal of the C-terminal dipeptide by angiotensin-I-converting enzyme (ACE), and additional detailed studies have shown that the actual immunodominant peptide is a decamer P18-I10 (RGPGRAFVTI) (Kozlowski et al., 1993). The effect of proteolytic processing on the antigenicity of P18IIIB peptide and its analogs was investigated by functional T cell assays based on the ability of T cell receptor (TCR) to recognize a specific major histocompatibility complex class I (MHC-I)/peptide complex. Recently we described a new monoclonal antibody (MAb) KP15 directed against the MHC-I molecule H-2Dd complexed with the 10-mer peptide P18-I10. Using this MAb, the cell surface H-2Dd/P18-I10 complex can be easily detected by flow cytometry (Polakova et al., 2000). Here we examined whether peptides longer than P18-I10 decamer form H-2Dd complexes recognized by KP15 MAb. Further we also analyzed how the ACE processing of P18IIIB-related peptides of different length affects their ability to form complexes with H-2Dd recognized by MAb KP15. These experiments confirmed that the ACE digestion of 15-mer peptide P18IIIB is the most effective in the production of a peptide capable of forming complex with H-2Dd recognized by KP15 MAb. The ACE digestion of longer peptides (16-mer to 19-mer) did not produce a significant quantity of peptides, capable of forming H-2Dd complexes recognizable with by MAb KP15. Peptides shorter than P18IIIB (13-mer to 10-mer), notably the optimally sized P18-I10 peptide lost most of their capacity to form H-2Dd complexes recognized by KP15 MAb. Our results show that the extracellular processing of MHC-I-restricted peptides, which cannot be overlooked in designing peptide-based vaccines, can be also studied by as simple and rapid assay as flow cytometry, provided MAbs specific to a particular MHC-I/peptide complex are available.     

Newly Characterized Species-Specific Immunogenic Chlamydophila pneumoniae Peptide Reactive with Murine Monoclonal and Human Serum Antibodies

A monoclonal antibody (MAb) directed against an unknown Chlamydophila pneumoniae epitope has been characterized, and the respective peptide mimotope has been identified. A murine MAb specific for C. pneumoniae was used to select peptides from phage display libraries. The peptides identified from the phage display library clones reacted specifically with the respective target murine MAb and with human sera previously identified as having antibody titers to C. pneumoniae. The selected peptide mimotope sequences tended to be composed of charged residues surrounding a core of hydrophobic residues. The peptide with the best binding could inhibit >95% of binding to the MAb, suggesting that the selected peptide binds the paratope of the respective MAb. The peptide reacted with human sera previously determined by microimmunofluorescence to have anti-C. pneumoniae antibodies. The peptide was competitively competed with the MAb against Renografin-purified, sonicated C. pneumoniae in an enzyme-linked immunosorbent assay and with whole-cell C. pneumoniae in an indirect fluorescence assay format, demonstrating its potential utility in the development of diagnostics. The use of this novel peptide may allow investigators to establish standardized assays free from cross-reactive Chlamydia trachomatis and Chlamydophila psittaci epitopes and immunoreactivity.     

The identification of peptide sequences of human chorionic gonadotropin containing a conformational epitope

A series of overlapping peptides were synthesized representing the entire amino acid sequence of the beta-subunit of human chorionic gonadotropin (hCG) and these were reacted with a monoclonal antibody shown to be specific for hCG. One linear peptide (residues 40-52 of the sequence) reacted significantly with the monoclonal antibody but a conjugate of this peptide to diphtheria toxoid (DT) failed to elicit significant levels of antibodies reactive to hCG in rabbits. The subsequent preparation of an extended peptide (residues 38-57) in which the two cysteines were oxidized to form a loop peptide yielded a highly immunogenic antigen when conjugated to DT. Antibody levels reactive with hCG from loop peptide immunizations of rabbits exceeded those found after immunization with a 37 residue peptide representing the carboxyl terminus of the beta-hCG subunit. The antisera did not react with pituitary glycoprotein hormones with similar sequences.