Prostate-specific antigen: characterization of epitopes by synthetic peptide mapping and inhibition studies

To improve our understanding of the prostate-specific antigen (PSA) antigenic regions, we studied the association targets of one anti-PSA polyclonal antibody and 10 anti-PSA monoclonal antibodies (mAbs). We also examined the ability of the mAbs to inhibit PSA enzymatic activity and block the association of PSA with alpha 1-antichymotrypsin (ACT). Linear epitope mapping with a polyclonal antibody indicated the presence of six major antigenic regions in PSA. Examination of the panel of mAbs established that three of them bind to linear epitopes. Five of the mAbs inhibited > 90% of PSA enzymatic activity. However, inhibition of PSA enzymatic activity and hindrance of PSA-ACT association by mAbs cannot be used to predict whether the mAbs bind to free PSA, the PSA-ACT complex, or both. Some of the mAbs may block PSA-ACT association through peripheral occlusion of the binding site, or through induction of conformational changes in PSA.     

Cross-reactivity of ten anti-prostate-specific antigen monoclonal antibodies with human glandular kallikrein

OBJECTIVES: Prostate-specific antigen (PSA) is commonly used as a marker for prostate disease. Prostate epithelium expresses both PSA and human glandular kallikrein (hK2) proteins, which share 80% sequence homology. The immunologic cross-reactivity of these two proteins could potentially interfere with determination of PSA levels in diagnoses of prostate cancer. We set out to determine the extent of this cross-reactivity for a panel of 10 anti-PSA monoclonal antibodies (mAbs). METHODS: Enzyme-linked immunosorbent assay (ELISA), sandwich assays, and western transfer techniques were used to assess the PSA/hK2 cross-reactivity of the anti-PSA mAbs. RESULTS: We did not detect the hK2 protein with any of the 10 anti-PSA mAbs under western transfer conditions. In ELISA experiments, 8 of 10 mAbs exhibited hK2 cross-reactivity under certain conditions. However, no combination of mAbs tested in sandwich assays exhibited a signal in hK2 cross-reactivity experiments greater than 0.1% of the PSA signal. CONCLUSIONS: We have evaluated 10 anti-PSA mAbs and determined that despite the 80% homology between PSA and hK2 proteins, cross-reactivity with hK2 by these antibodies would not significantly affect the determination of PSA levels by means of sandwich assays.     

Characterisation of epitopes of type 1 fimbriae of Salmonella using monoclonal antibodies specific for SEF21 fimbriae of Salmonella enteritidis

Monoclonal antibodies (mAbs) were used to identify and characterise epitopes of type 1 (SEF21) fimbriae of Salmonella enteritidis. The distribution of the epitopes among salmonellas and other enterobacteria was investigated, as well as the influence of growth media and temperatures on their expression. At least four different epitope clusters were identified on SEF21 fimbriae of S. enteritidis. Two of these clusters were associated with fimbrial haemagglutinins that were either common to all salmonellae tested, or restricted only to S. enteritidis and S. dublin. The four epitope clusters were identified on type 1 fimbriae of most Salmonella serotypes, as well as non-haemagglutinating type 2 fimbriae of S. pullorum and S. gallinarum, and on many other enterobacterial species. The expression of the epitopes was affected by growth conditions.     

Human single-chain Fv antibodies to MUC1 core peptide selected from phage display libraries recognize unique epitopes and predominantly bind adenocarcinoma

The tumor-associated antigen MUC1 is overexpressed and underglycosylated in human adenocarcinomas of diverse origins, such as breast, ovary, and colon. We isolated and describe five human single-chain (sc) Fv antibodies specific for the MUC1 variable number of tandem repeats region isolated by in vitro selection from a large naive phage antibody library containing over 6 x 10(9) different scFv antibodies. A synthetic biotinylated 100-mer peptide corresponding to five tandem repeats of the MUC1 peptide core was used for selection. Two of the antibodies were highly specific for MUC1 as judged by ELISA and flow cytometry. In immunohistochemistry, antibody clone 10A stained MUC1 in the cytoplasm and membrane of adenocarcinoma cells of breast and ovary, whereas in normal epithelium, only cytoplasmic or no staining was observed. With antibody clone 10B, staining was less pronounced and was not always membrane associated in adenocarcinoma. Determination of the fine specificity of 10A and 10B using a novel "indirect epitope fingerprinting" ELISA showed that both antibodies recognize unique epitopes that have not been described for hybridoma-derived anti-mucin antibodies of mouse origin. The selected human antibodies, like many of the murine MUC1 antibodies, recognize epitopes on the protein core of MUC1 that are abundantly present in the underglycosylated form of cell surface mucin on adenocarcinoma. The best human scFv, clone 10A, appears to distinguish normal cells from adenocarcinoma cells, which makes it an attractive candidate for use in antibody-based tumor targeting.     

Peak expiratory flow rate in school-going children

Two members of a unique class of natural antibodies have been identified in all of a large cohort of sera from clinically normal humans of broad age distribution. By means of a series of 10-12 mer peptides the epitope for each of those antibodies was characterized with regard to amino acid identity and conformation. Similar epitope specificity was revealed for the IgM isotopes of cord blood and early post natal sera and for IgM and IgG of adult sera, suggesting that the class of natural antibodies represented by the two identified in this study includes those genomically coded for at their effector level of maturation in the B cells of the neonate. Assay of series of specimens from each of four clinically normal adults revealed that those two natural antibodies are present at relatively constant titer, unique to each individual, over four to five and a half year periods. Those observations imply that the primary function of that class of natural antibodies may be related to maintenance of homeostasis and the molecular identity of each of the two epitopes suggests a role, for each, as monitor or control in intracellular traffic. The previous identification of those epitopes in a conserved protein of HIV also provides support for the proposition that a secondary function of natural antibodies, arising from fortuitous coincidence of the identity of the epitopes, may be that of early defense against infectious invaders.     

Prediction of epitopes and production of monoclonal antibodies against gastric H,K-ATPase

Monoclonal antibodies (mAbs) were produced against gastric H,K-ATPase using a theoretical and experimental strategy based on prediction of linear epitopes by molecular modelling followed by production of anti-peptide antibodies. By analysing the alpha subunit sequence, we predicted several epitopes corresponding to amino acids K519-L533, E543-Y553 and S786-L798 and produced monoclonal antibodies HK519, HK543 and HK786. All three react against gastric H,K-ATPase in RaLISA, immunohistochemistry and Western blots demonstrating that they recognize the native and the SDS-denatured ionic pump and that the epitopes are located at the surface of the native ATPase. Antibody Kd are in the range 6-10x10(-8) M. Monoclonal antibody HK519 is a competitive inhibitor of ATP, in agreement with ATP binding to K519. Neither mAb 543, nor mAb 786 inhibit the ATPase activity. Monoclonal antibody 95111, whose epitope is mapped between residues C529 and E561, competes with mAb HK543 but not with the other two. We suggest that the 95111 epitope is overlapping or very close to the HK543-553 sequence. Induction of E1 conformer by binding FITC to K519 increases the number of mAb 95111 and mAb HK543 epitopes but not that of mAb 786, supporting the fact that the fragment E543-Y553 changes accessibility, maybe during the E1-E2 transconformation.     

Definition of the specificity of monoclonal antibodies against porcine CD45 and CD45R: report from the CD45/CD45R and CD44 subgroup of the Second International Swine CD Workshop

Swine cell binding analyses of a set of 48 monoclonal antibodies (mAbs), including eleven standards, assigned to the CD44 and CD45 subset group of the Second International Swine CD Workshop yielded 13 clusters. Although none of these corresponded to CD44, seven mAbs formed a cluster which was identified as being specific for restricted epitopes of CD45 (CD45R). In addition, a T-cell subset specific cluster comprised of four mAbs was also identified. Two mAbs (STH106 and SwNL 554.1) reacted exclusively with CD8 bright lymphocytes, the other two (2B11 and F01G9) with a subset of CD4 lymphocytes. The other 10 clusters were either specific for MHC-class I like molecules or overlapped with clusters identified by the adhesion molecule subgroup and are therefore just briefly discussed in this report. The specificity of all the mAbs in the CD45R cluster was verified by their ability to immunoprecipitate distinct proteins and to react with CHO cells expressing individual isoforms of CD45. Three CD45R mAbs (3a56, MIL5, -a2) did react with a 210 kDa isoform(s), while another three (STH267, FG2F9, 6E3/7) only recognized a 226 kDa isoform(s). The remaining one (MAC326) precipitated both a 210 and 226 kDa protein. The specificity of all the mAbs in the CD45R cluster, and of the CD45 common mAbs, was confirmed by their reactivity with CHO cells transfected with cDNAs encoding the extracellular and transmembrane portions of distinct CD45R isoforms. Those mAbs recognizing a 210 kDa protein reacted with CHO cells expressing the CD45RC isoform, while those capable of precipitating a 226 kDa, but not the 210 kDa, polypeptide recognized CHO cells expressing either the CD45RAC and the relatively rare CD45RA isoform. MAC326 was unique in its inability to react with CHO cells engineered to produce the CD45RC and CD45RAC isoforms. Thus, three mAbs (6E3/7, STH267, and FG2F9) appear to be specific for an epitope(s) encoded by the A exon, while one (MAC326) recognizes a determinant encoded by the C exon. The remaining three mAbs (3a56, -a2, MIL5) are apparently specific for an epitope(s) which results from the fusion of the C exon to the invariant leader sequence and is destroyed by inclusion of the A exon. All three CD45 common mAbs, K252.1E4, MAC323 and 74.9.3, did react with the CHO cells lines expressing either the CD45RA, CD45RC, CD45RAC or CD45RO isoforms, but not with untransfected CHO cells. When the natural expression of CD45 isoforms was examined by reacting lymphocytes with CD45R mAbs, a high level expression of isoforms containing the A exon-generated domain was detected in all B cells while the majority of CD4+ T cells had undetectable or lower expression density of this protein than B cells. In contrast, the density of expression of the CD45 isoform(s) containing the C exon-generated domain ranged from undetectable to high in CD4+ T cells whereas the amounts were approximately ten-fold lower in B cells. Overall this panel of CD45 mAbs will be very useful in analyzing the maturation and differentiation of swine lymphoid cells subsets.     

Obstetricians'' knowledge and attitudes toward epidural analgesia in labour

A 30 kDa immunodominant surface antigen (p30) of Babesia equi has been used as a diagnostic antigen. The B cell epitopes on this molecule recognized by horse sera and monoclonal antibody (MAb) against p30, 36/133.97, were determined. A synthetic peptide of p30 with amino acid sequence of 123FYQEVLFKGFEAV135 exhibited strong positive reaction with the infected horse sera. In contrast, MAb 36/133.97 recognized different region of p30, as peptide synthesized with amino acid sequence of 27ASGAVVDFQLESI39 reacted strongly. In competitive inhibition ELISA, the binding of MAb 36/133.97 to recombinant p30 was inhibited by horse antibodies, although they did not recognize same or an overlapping epitope. The data on B cell epitopes in this study may be important in improving serodiagnostic methods of B. equi infection.     

The humoral immune response to human T-cell lymphotropic virus type 1 envelope glycoprotein gp46 is directed primarily against conformational epitopes

Individuals infected with human T-cell lymphotropic virus type 1 (HTLV-1) develop a robust immune response to the surface envelope glycoprotein gp46 that is partially protective. The relative contribution of antibodies to conformation-dependent epitopes, including those mediating virus neutralization as part of the humoral immune response, is not well defined. We assess in this report the relationship between defined linear and conformational epitopes and the antibodies elicited to these domains. First, five monoclonal antibodies to linear epitopes within gp46 were evaluated for their ability to abrogate binding of three human monoclonal antibodies that inhibit HTLV-1-mediated syncytia formation and recognize conformational epitopes. Binding of antibodies to conformational epitopes was unaffected by antibodies to linear epitopes throughout the carboxy-terminal half and central domain of HTLV-1 gp46. Second, an enzyme-linked immunoadsorbent assay was developed and used to measure serum antibodies to native and denatured gp46 from HTLV-1-infected individuals. In sera from infected individuals, reactivity to denatured gp46 had an average of 15% of the reactivity observed to native gp46. Third, serum antibodies from 24 of 25 of HTLV-1-infected individuals inhibited binding of a neutralizing human monoclonal antibody, PRH-7A, to a conformational epitope on gp46 that is common to HTLV-1 and -2. Thus, antibodies to conformational epitopes comprise the majority of the immune response to HTLV-1 gp46, and the epitopes recognized by these antibodies do not appear to involve sequences in previously described immunodominant linear epitopes.     

Mapping of conformational B cell epitopes within alpha-helical coiled coil proteins

An approach to mapping antigenic B cell epitopes within alpha-helical coiled coil proteins has been developed and applied to two proteins: Streptococcal M protein and C. elegans paramyosin protein UNC-15. Overlapping peptides derived from an alpha-helical coiled coil conformational epitope were embedded between helical flanking peptides derived from the completely unrelated GCN4 leucine zipper peptide. The resulting chimeric peptides exhibited helical propensity. Chimeric peptides were tested for antigenicity (recognition by antibody) or immunogenicity (production of appropriate antibody response). A conformational epitope within the Streptococcal M protein recognised by three mAbs spanned 12 residues. Analysis of chimeric peptides based on C. elegans UNC-15 has enabled fine mapping of the minimal B cell epitope recognised by monoclonal antibody NE1-6B2 to seven non-contiguous residues (spanning 15 residues); the footprint of contact residues involved in antibody recognition being restricted to the hydrophilic face of the helix and covering five helical turns. This chimeric peptide epitope when coupled to diphtheria toxoid was highly immunogenic in mice and antisera recognised the conformationally dependent native peptide epitope. This approach has the potential to map conformational epitopes and design minimal epitopes for use as vaccine candidates.     

A role for polysialic acid in neural cell adhesion molecule heterophilic binding to proteoglycans

The neural cell adhesion molecule (NCAM) is known to participate in both homophilic and heterophilic binding, the latter including mechanisms that involve interaction with proteoglycans. The polysialic acid (PSA) moiety of NCAM can serve as a negative regulator of homophilic binding, but indirect evidence has suggested that PSA can also be involved in heterophilic binding. We have examined this potential positive role for PSA in terms of the adhesion of PSA-expressing mouse F11 cells and chick embryonic brain cells to substrates composed of the purified heparan sulfate proteoglycans agrin and 6C4. This adhesion was specifically inhibited by polyclonal anti-NCAM Fab antibodies, monoclonal anti-PSA antibodies, PSA itself, and enzymatic removal of either PSA or heparan sulfate side chains. By contrast, the adhesion was not affected by chondroitinase, and cell binding to laminin was not inhibited by any of these treatments. A specific NCAM-heparan sulfate interaction in this adhesion was further indicated by its inhibition with monoclonal anti-NCAM Fab antibodies that recognize the known heparin-binding domain of NCAM and with the HBD-2 peptide derived from this region, but not with antibodies directed against other regions of the protein including the homophilic binding region. Together, the results suggest that PSA can act in vitro either as a receptor in NCAM heterophilic adhesion or as a promoter of binding between heparan sulfate proteoglycans and the NCAM heparin-binding domain.     

Epitope polarity and adjuvants influence the fine specificity of the humoral response against Semliki Forest virus specific peptide vaccines

The humoral response to synthetic peptide vaccines against Semliki Forest virus (SFV) in H-2d BALB/c mice was investigated with the enzyme linked immunosorbent assay and the pepscan technique. The peptide vaccines consisted of amino acid sequences 240-255 (B) and 137-151 (T) of the E2 membrane protein of SFV colinearly synthesized in either orientation T-B or B-T. Sequence B contains an epitope inducing humoral immunity to lethal SFV infection and sequence T contains a H-2d restricted T-helper cell epitope. With sera from mice immunized subcutaneously with peptide T-B, and Quil A as adjuvant, two immunodominant B-cell epitopes were identified, FVPRAD, at position 240-246 and PHYGKEI, at position 145-151. However, with peptide B-T and Quil A as adjuvant for immunization the epitope PHYGKEI was clearly immunodominant, but antibodies elicited against this epitope were not reactive with SFV-infected L cells in contrast to the antibodies elicited by epitope FVPRAD. An additional epitope EPARKGKVH, at position 247-255, was identified with sera from mice immunized subcutaneously with either peptide T-B or B-T and Montanide ISA 740 as an adjuvant. Monoclonal antibodies selected for reactivity with SFV-infected L cells did bind also to epitope FVPRAD. Interestingly, this epitope could induce antibodies cross-reactive with a synthetic peptide derived from macrophage migration inhibitory factor that shares amino acid residues VPRA at position 9-12 with the protective B-cell epitope FVPRAD. The present study shows clearly that the fine specificity of the humoral response against peptide vaccines is differentially influenced by both adjuvant and epitope polarity which may affect vaccine efficacy. Further, the study reminds us that potentially autoimmune antibodies could be induced by vaccines.     

Antigenicity and immunogenicity of multiple antigen peptides (MAP) containing P. vivax CS epitopes in Aotus monkeys

Using linear synthetic peptides corresponding to the Plasmodium vivax circumsporozoite (CS) protein of the common type, we have identified several T and B-cell epitopes recognized by human individuals. Three T-cell epitopes studied (p6) from the amino, (p11) from the central and (p25) from the carboxyl regions, were widely recognized by lymphocytes of immune donors. A series of six peptides, in addition to p11, representing the central repeat domain of the CS(p11-p17) protein were used in ELISA assays to map the B-cell epitopes of this region. P11 was the peptide most frequently recognized by sera containing antibodies to the homologous CS protein as determined by IFAT. The sequences corresponding to peptides p6, p11 and P25 as well as that representing a universal T-cell epitope derived from the tetanus toxin were used to assemble eight different Multiple Antigen Peptides (MAP). The immunogenicity of these MAP was analysed in Aotus monkeys. Groups of two animals were immunized with each MAP and both antibody response, T-lymphocyte proliferation and in vitro gamma-IFN production were evaluated. Two MAPs containing the same B-cell epitope and either a promiscuous CS-protein derived T-cell epitope (p25) or the tetanus toxin epitope (p-tt30) proved to be the most immunogenic and induced high levels of anti-peptide antibodies that recognized the native protein. Except for animals immunized with MAP VII, there was no correlation between antibody levels, lymphocyte proliferation or gamma-IFN production in vitro. The broad recognition of these epitopes by individuals which had been exposed to malaria, the capacity of these MAPs to induce antibodies, recognize the cognate protein, and in vitro gamma-IFN production encourages further analyses of the potential of these proteins as malaria vaccine candidates for human use.     

Human glutamate dehydrogenase is immunologically distinct from other mammalian orthologues

Five monoclonal antibodies (mAbs) that recognize human glutamate dehydrogenase (GDH) have been selected and designated as monoclonal antibodies hGDH60-6, hGDH60-8, hGDH63-10, hGDH63-11, and hGDH91-14. A total of five mAbs recognizing different epitopes of the enzyme were obtained, two of which inhibited human GDH activity. When total proteins of human homogenate separated by SDS- PAGE, were probed with mAbs, a single reactive protein band of 55 kDa, which co-migrated with purified recombinant human GDH was detected. When the purified GDH was incubated with each of the mAbs, its enzyme activity was inhibited by up to 58%. Epitope mapping analysis identified, two subgroups of mAbs recognizing different peptide fragments. Using the individual anti-GDH antibodies as probes, the cross reactivities of brain GDH obtained from human and other animal brain tissues were investigated. For the human and animal tissues tested, immunoreactive bands on Western blots appeared to have the same molecular mass of 55 kDa when hGHD60-6, hGHD60-8, or hGHD91-14 mAbs were used as probes. However, the anti-human GDH mAbs immunoreactive to bands on Western blots reacted differently on the immunoblots of the other animal brains tested, i.e., the two monoclonal antibodies hGDH63-10 and hGDH63-11 only produced positive results for human. These results suggest that human brain GDH is immunologically distinct from those of other mammalian brains. Thorough characterization of these anti-human GDH mAbs could provide potentially valuable tool as immunodiagnostic reagents for the detection, identification and characterization of the various neurological diseases related to the GDH enzyme.     

Measurements of colour constancy by using a forced-choice matching technique

In order to compare the reactivity of glutamic acid decarboxylase (GAD) antibodies recognizing linear and conformational epitopes as islet cell cytoplasmic antibodies (ICA), monoclonal antibodies were generated. An ELISA displacement test using two biotinylated monoclonals recognizing a linear (M61/7E11) or a conformational GAD65 epitope (M65/6B12) was performed to identify epitope regions recognized by monoclonal GAD antibodies. The GAD binding by monoclonal GAD antibodies was tested by immunofluorescence on fixed and unfixed pancreatic sections of human, rat, and mouse, and by Dot-blot experiments. 16/23 (69.6%) of the monoclonals were specifically reactive with GAD65 and 7/23 (30.4%) were reactive with both GAD isoforms. 8/16 (50%) of monoclonal GAD65 antibodies recognized a linear GAD epitope located at the N-terminus (pattern 1). 5/16 (31.3%) displaced M65/6B12, indicating the recognition of a conformational GAD epitope (pattern 2). Monoclonals belonging to patterns 1 and 2 showed strong ICA binding. 3/16 (18.8%) of monoclonals specific for GAD65 with weak or no immunostaining of pancreatic islets (pattern 3) did not inhibit the binding of both biotinylated antibodies in the displacement test, indicating other epitope specificities. In conclusion, GAD antibodies recognizing both conformational and linear epitopes of the GAD65 molecule are involved in ICA binding with strong reactivity. Furthermore, results obtained with monoclonals of pattern 3 suggest the occurrence of GAD65 epitopes partly inaccessible on cryosections, which may result in an ICA-negative test of GAD65 autoantibody positive sera.     

Epitope mapping of T4 endonuclease VII with monoclonal antibodies reveals importance of both ends of the protein for target binding

Endonuclease VII (endo VII) of bacteriophage T4 is a Holliday-structure resolving enzyme that can also recognize many other defects in DNA via an altered secondary structure. The protein has a molecular mass of 18 kDa and exists as a dimer in solution. Here we report the production and characterization of monoclonal antibodies (mAbs) directed against the highly purified enzyme. From one fusion 15 hybrid cell lines producing mAbs with high affinity for endo VII could be established. The mAbs were used for epitope mapping of the protein by using N-terminal, C-terminal and internal peptides of endo VII as antigens in enzyme-linked immunoabsorbant assays. Three classes of mAbs were distinguished as follows: (1) the predominant class with 13 mAbs recognized a C-terminal epitope located between amino acid residues 115 and 145; (2) a second class, represented by one mAb, recognized an epitope located at the N terminus between amino acid residues 16 and 65; (3) a third class, represented by one mAb, recognized an epitope built from nearly the entire native protein including amino acid residues from the C and N terminus of endo VII. The latter finding suggests close proximity of the two ends, which are provided apparently by the same monomer, since the mAb from class III does also react with a mutant protein deficient in dimerization. Internal sequences of endo VII between amino acid residues 78 and 145 did not react with any of the mAbs.     

Sensitization of rat glioblastoma multiforme to cisplatin in vivo following restoration of wild-type p53 function

A panel of seven monoclonal antibodies (mAbs) raised against cardiac troponin-I (CdTnI) isolated from canine and human hearts, which have been shown to be cardiac-specific but cross-species reactive [Cummins, B., Aukland, M. L. & Cummins, P. (1987) Amer. Heart J. 113, 1333-1344], were used in this study. These mAbs were tested against recombinant wild-type and mutant human CdTnI proteins to assess their value as probes for the phosphorylation status of CdTnI. Four mAbs were found to react positively with the recombinant wild-type protein and their epitopes were contained in residues 31-210 of the human cardiac protein. Two of these mAbs appeared to be directed against the same epitope site within this region. The remaining three mAbs only reacted against the recombinant wild-type protein when it was phosphorylated, showing that these three antibodies were directed against the phosphate group(s) on Ser23 and/or Ser24. In order to investigate this further, a series of single and double mutants of CdTnI were used in which either Ala (to direct the enzymatic phosphorylation) or Asp (to mimic the phosphate group) replaced the Ser23 and/or Ser24. It was found the all three mAbs were able to react with the mono-phosphorylated form of the [Ala23]CdTnI single mutant but not the mono-phosphorylated form of the [Ala24]CdTnI single mutant, showing that they specifically required phosphorylation at Ser24. Experiments with a synthetic peptide composed of residues 1-29 of human CdTnI confirmed these data. Two of the three phosphorylation-specific mAbs were able to react with mutants containing either two Asp residues replacing Ser23 and Ser24 or one Asp residue instead of Ser24, indicating that a negative charge at position Ser24 is sufficient to invoke a reaction. The other mAb was more specific in that it would only react with CdTnI species with a phosphate group on Ser24.     

Delineation of tyrosine-containing epitopes within the beta subunit of bovine thyrotropin

The epitopes recognised by two monoclonal antibodies (mAb 279 and mAb 299), specific for the beta subunit of bovine thyroid-stimulating hormone (bTSH), have been localised using a technique in which the tyrosine residues in the bTSH beta subunit were subjected to modification when the bTSH beta subunit was complexed with either mAb or in the free, unbound state. The epitope recognised by mAb 279 was localised to the C-terminal region of bTSH beta with the tyrosine residue Tyr104 protected from modification by the presence of this mAb. In addition, the experimental results indicate that the tyrosine residues Tyr18 and/or Tyr112 are also involved in the mAb 279 epitope. The epitope recognised by mAb 299 was localised to the region 59-74 of bTSH beta as both Tyr59 and Tyr74 were protected from modification by the presence of this mAb. Since both mAbs have been previously found to inhibit receptor binding, the sequence regions/amino acid positions recognised by these mAbs are likely to represent determinants for receptor binding. Moreover, these data indicate that the identified amino acid residues are located on the surface of the molecule, consistent with predictions of the tertiary structure of the bTSH beta subunit based on the recently elucidated X-ray crystal structure of human chorionic gonadotropin.     

Criminality in a sample of drug abusers in Greece

In order to produce mAbs directed specifically against HLA-DR10 molecule, transfected mouse L cells, expressing the DRB1*1001 allele, were used to immunize C3H mice over a period of 4 weeks. Two mAbs, 2C12 and 4B6, derived from this fusion were found to recognize, with different affinity, polymorphic epitopes of DR10 that are shared with DR1, 3, 7, and 9. These mAbs were screened on a large panel of homozygous B lymphoblastoid cell lines using microlymphocytotoxicity and the results were confirmed by flow cytometry. The reactive pattern of 2C12 and 4B6 was compared to that of MP10 human mAb also recognizing the DR10 specificity in addition to DR1, 2 and 9. Based on serologic specificity and cellular absorption experiments, we conclude that the epitopes the murine and human mAbs respectively recognize on the DR10 molecule, are probably different.     

Molecular characterization of two monoclonal antibodies specific for the LDL receptor-binding site of human apolipoprotein E

Apolipoprotein E (apoE), a 299 amino acid protein, is a ligand for the low density lipoprotein receptor (LDLr). It has been established that basic amino acids situated between apoE residues 136 and 150 participate in the interaction of apoE with the LDLr. Evidence suggests that apoE is heterogeneous on lipoproteins in its conformation and in its ability to react with cell surface receptors. Our goal was to produce mAbs that could serve as conformational probes of the LDLr binding site of apoE. We used a series of apoE variants that have amino acid substitutions at residues 136, 140, 143, 144, 145, 150, 152, and 158 to identify the epitopes of two anti-human apoE monoclonal antibodies (mAbs), 1D7 and 2E8, that inhibit apoE-mediated binding to the LDLr. We show that most of the variants that have reduced reactivity with the LDL receptor also have reduced reactivity with the mAbs. The epitopes for both mAbs appear to include residues 143 through 150 and thus coincide with the LDLr-binding site of apoE. It is notable that mAb 2E8, but not 1D7, resembles the LDLr in showing a reduced reactivity with apoE (Arg158 --> Cys). While most of the receptor-defective variants involve replacement of apoE residues directly implicated in binding, substitution of Arg158 by Cys is thought to indirectly affect binding of apoE to the LDLr by altering the conformation of the receptor-binding site. To determine whether the similarity in specificities of the mAbs and the LDLr reflect structural similarities, we cloned and characterized the cDNAs encoding the light and heavy chains of both mAbs. Primary sequence analysis revealed that, although these two antibodies react with overlapping epitopes, their respective complementarity determining regions (CDRs) share little homology, especially those of their heavy chains. The two mAbs, therefore, likely recognize different epitopes or topologies within a limited surface of the apoE molecule. Four negatively charged amino acids were present in the second CDR of the 2E8 heavy chain that could be approximately aligned with acidic amino acids within the consensus sequence of the LDLr ligand-binding domain. This could indicate that mAb 2E8 and the LDLr use a common mode of interaction with apoE.