Monoclonal Antibody-defined Epitopes Research Articles

The Clinical Utility of Tumor Markers

Tumor markers provide a minimally invasive, cost-effective source of data valuable for monitoring disease course, determining prognosis, and aiding in treatment planning. An understanding of the individual test characteristics and limitations, however, is important for optimal use and accurate interpretation of results. A variety of types of markers are available in the routine clinical laboratory including enzymes, oncofetal antigens, oncogene products, monoclonal antibody-defined epitopes, and hormones. The clinical applications and limitations of some that are currently in widespread use will be discussed.

A family of phase- and size-variant membrane surface lipoprotein antigens (Vsps) of Mycoplasma bovis

A set of strain- and size-variant highly immunogenic membrane surface protein antigens of Mycoplasma bovis, which has been identified by a monoclonal antibody, is shown in this report to make up a family of antigenically and structurally related lipid-modified proteins, designated Vsps (variable surface proteins). By systematic analysis of several isogenic clonal lineages of the type strain PG45, three members of this family have been identified, VspA, VspB, and VspC, each of which was shown to undergo independent high-frequency changes in size as well as noncoordinate phase variation between ON and OFF expression states. The monoclonal antibody-defined epitope common to VspA, VspB, and VspC was accessible on the cell surface in most, but not all, of the clonal populations analyzed and was present on a C-terminal limit tryptic fragment of each Vsp variant that was released from the membrane surface. VspA and VspC were distinguished from VspB by their selective detection with colloidal gold and by their distinctive reaction with a polyclonal antibody against M. bovis D490. VspA, VspB, and VspC were further distinguishable from one another by their characteristic patterns of degradation at carboxypeptidase Y pause sites. While these Vsp-specific structural fingerprints with an irregular periodic spacing were constant for similarly sized variants of a defined Vsp product, they showed distinct differences among variants differing in size. This variability included gain or loss of individual bands within distinct subsets of bands, as well as shifts of the entire banding patterns up- or downwards, indicating that insertions or deletions underlying Vsp size variation can occur at various locations either within the C-terminal domain or within other regions of these proteins. This was similarly confirmed by comparative epitope mapping analysis of tryptic cleavage products generated from different Vsp size variants. The Vsp family of M. bovis described in this study represents a newly discovered system of surface antigenic variation in mycoplasmas displaying features which closely resemble but are also different from the characteristics reported for the Vlp (variable lipoprotein) system of M. hyorhinis. The isogenic lineages established here provide key populations for subsequent analysis of corresponding genes to further elucidate Vsp structure and variation, which may have important relevance for a better understanding of the pathogenicity of this agent.

Interactions Between Zinc and Thymulin

Thymulin (formerly called "Facteur Thymique Sérique or FTS) is a metallopeptidic hormone selectively produced by thymic epithelial cells (TEC) and known to induce intra and extra-thymic T cell differentiation. It was initially isolated from porcine serum and shown to be present in calf thymus extract. Its amino-acid sequence was determined (<Glu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn). It is a nonapeptide whose biological activity is dependent on the presence of zinc, in an equimolecular ratio. The metallopeptide thus formed bears a specific tridimensional conformation detected by nuclear magnetic resonance studies, and that yielded a new monoclonal antibody-defined epitope. The presence of zinc and metallothionein has been demonstrated within TEC which produce the peptide, suggesting that the molecule is secreted in its active zinc-containing form. The zinc/thymulin relationship, was further studied using various models of mild zinc deficiency in experimental animals and in humans. Serum thymulin activity was decreased as a result of zinc deficiency, and was corrected by in vivo and in vitro zinc supplementation, suggesting that this parameter could be a sensitive indicator of zinc deficiency. When considered together with the parallel variations seen in T-cell subpopulations and lymphokine production, these observations could provide a possible explanation of the role of zinc on T cell functions.

The Immune Response of Children to Meningococcal Lipooligosaccharides during Disseminated Disease Is Directed Primarily against Two Monoclonal Antibody-Defined Epitopes

A human inhibition monoclonal ELISA (HIMELISA) was used to investigate the immune response of infants and children to meningococcal lipooligosaccharide (LOS). Convalescence from disseminated meningococcal disease significantly increased the inhibition by sera of monoclonal antibody (MAb) binding to two of six defined epitopes on the LOS of meningococcal strain 126E, a strain previously shown to express immunogenic LOS epitopes. The inhibited epitopes were defined by MAbs D6A and 6B7, and both were expressed on the 3.6-kDa LOS of strain 126E. The inhibition of the binding of both MAbs by the convalescent sera was similar to that from children who were meningococcal carriers and greater than that by sera obtained from healthy children. These results support the conclusion that the 3.6-kDa LOS molecule of strain 126E expresses two conserved epitopes that are immunogenic in infants and children; this LOS may serve as a vaccine candidate.

In vitro mutagenesis of HLA-B27: Single and multiple amino acid substitutions at consensus B27 sites identify distinct monoclonal antibody-defined epitopes

the consensus HLA-B27 sequence includes a unique constellation of amino acid residues along the peptide-binding cleft. To investigate the potential role of this region in the antigenic structure of HLA-B27, a panel of transfected cell lines was produced expressing 24 mutant B27 molecules with single or multiple susbtitutions within this constellation of residues. The cells were analyzed by flow cytometry with a panel of four anti-B27 mAb; ME1, GSP5.3, GS145.2, and B27M2. Previous studies have suggested that position 67 exerts a conformational effect on the ME1, GSP5.3, and GS145.2 epitopes. This was further supported in these studies by the observation that additional substitutions at the flanking residues 63 and 70 could reverse the disruption of these mAb epitopes by large residues at 67. Substitutions at positions 69–71 disrupted the binding of ME1 and GSP5.3, apparently by a direct effect. Individual at either of the two positions bearing residues unique to B27, 70 and 97, had no significant influence on the binding of any of the four mAb. The region of amino acid positions 63–71 in HLA-B27 thus appears to participate in the formation of at least three distinct epitopes shared by B27 and B7, identified by ME1, GSP5.3, and GS145.2.

Monoclonal antibody-defined epitope map of expressed rubella virus protein domains

An expanded library of murine monoclonal antibodies (MAbs) was generated by infecting BALB/C mice with the Therien strain of rubella virus (RV) and selecting secreting hybrids by enzyme-linked immunosorbent assay (ELISA) using purified virion targets. A panel of plasmids containing specified RV cDNA fragments was also constructed by using a variety of strategies with pGE374- and pGE374-derived expression vectors. Hybrid RecA-RV-beta-galactosidase (LacZ)- or RecA-RV-truncated LacZ-containing proteins collectively representing the entire open reading frame of the structural proteins of RV were overexpressed in Escherichia coli. Bacterial lysates were then probed by ELISA with selected MAbs and by immunoblot following separation by electrophoresis under denaturing conditions. With this approach, MAbs that appeared to react with linear determinants defined epitopes localized within the following domains: MAbs C-1, C-2, and C-8 bind epitopes within the predicted amino-terminal 21 amino acids of the capsid region C9 to C29; MAb C-9 binds to a domain bounded by C64 and C97; MAbs E2-1 through E2-6 bind to the E2 glycoprotein backbone region from E2(1) to E2(115); MAbs E1-18 and E1-20 bind to the E1 glycoprotein region from E1(202) to E1(283). MAb E1-18 neutralizes RV infectivity; MAb E1-20 neutralizes infectivity and modestly inhibits hemagglutination. Analyses with selected synthetic peptides have confirmed several of the molecular domains deduced with the expressed proteins. These plasmid constructions and peptides have proven useful in beginning to unravel the molecular organization of several antigenic sites of this human pathogen.

Stability of monoclonal antibody-defined epitopes

Epitope instability can limit the applications of monoclonal antibody (mAb) technology in laboratory and clinical research. We exposed a group of representative antigens on human hepatocellular carcinoma (HCC) cells to physiochemical insults to study epitope stability as measured by mAb immunoreactivity. Each epitope was found to have a unique pattern of instability which serves to biophysically characterize the antigen and defines the conditions to which the antigen can be exposed during laboratory and clinical investigations. Individual antigens were found to be unstable within a surprisingly well defined window of solvent polarities while being stable on either side of that window. Several antigens were observed to be unstable when exposed to transient changes in pH. When a critical temperature between 42° C and 65° C was achieved, epitopes which were thermosensitive underwent a sudden loss in immunoreactivity. This critical temperature was found to be pH dependent. The effects of polarity, pH, and temperature on epitope stability are consistent with changes in protein structural conformation. In addition, we found that certain fixatives cause a time and concentration dependent loss of epitope immunoreactivity. This study provides a rapid and easy determination of monoclonal antibody-defined epitope stability; the results of which serve to guide further studies on the antigen and to characterize the antigen on the basis of its unique physiochemical stability.

Characterisation of an inhibitory monoclonal antibody-defined epitope on a malaria vaccine candidate antigen

A monoclonal antibody that recognizes a recently characterised 45-kDa merozoite surface antigen of the human malaria parasite Plasmodium falciparum inhibits the growth of the asexual blood stages of the parasite in vitro. The corresponding epitope has been determined by testing the reactivity of the antibody with sequentially overlapping octapeptides. A synthetic peptide containing the epitope elicits antibodies that react with the native antigen. Epitope mapping in this manner is useful in the design of synthetic vaccines against malaria.

Structural studies on a putative Plasmodium knowlesi merozoite antigen

Two monoclonal antibodies, which prevent merozoites attaching to or invading erythrocytes, react with the same or closely apposed epitopes on a minor 66 kDa Plasmodium knowlesi antigen. The antigen is processed, at the time of schizont rupture and merozoite release, to 44 and 42 kDa molecules which are present on the merozoite surface [Deans, J.A. et al. (1984) Mol. Biochem. Parasitol. 11, 189–204]. The monoclonal antibody-defined epitope, which is expressed only once on the 66 kDa molecule, is formed by a tertiary folding of the polypeptide chain (minimum size 42 kDa). The conformation of the epitope is maintained by weak intramolecular forces of attraction, rendering the epitope extremely labile; it is completely destroyed by treatment with sodium dodecyl sulphate (SDS). Polyclonal monospecific antiserum raised against SDS-treated antigen did not inhibit parasite proliferation whereas a polyclonal antiserum raised against native antigen was inhibitory. It is postulated that the monoclonal antibody-defined antigenic determinant is crucial for merozoite invasion.

Mapping of monoclonal antibody-defined epitopes associated with carcinoembryonic antigen, CEA.

Six immunoglobulin G monoclonal antibodies reactive with carcinoembryonic antigen (CEA) were evaluated with respect to parameters implicated in their potential diagnostic application and use as tumor targeting agents for cytotoxic drugs or plant or bacterial toxins. Antibody reactivity with surface antigens of the MKN-45 gastric tumor cell line was demonstrated by flow cytofluorimetry. In a subcellular membrane binding assay, each antibody reacted preferentially with membranes isolated from colorectal tumor tissue in comparison with their reaction with membranes from adjacent, apparently normal colonic mucosa. Three of the antibodies (NCRC-23, C228, and 11.285.14) reacted specifically with CEA with little or no reaction with the cross-reacting antigen, NCA. The remaining three antibodies (C24, C161, and C198) were reactive with both CEA and NCA. Analysis of the epitopes defined by these antibodies was performed by competitive binding inhibition assays evaluating the capacity of unlabeled antibodies to compete with 125I-labeled antibodies in their binding to CEA. In addition, double determinant or 'sandwich' radioimmunoassays were employed to examine the coexpression of epitopes on CEA molecules. These studies permitted an epitope map to be constructed which describes the coincidence, overlapping, or independent expression of both CEA specific epitopes and epitopes shared between CEA and NCA. The map may be employed for the selection of antibodies for diagnostic and therapeutic use.

Immunochemical characterization and purification of Sm-97, a Schistosoma mansoni antigen monospecifically recognized by antibodies from mice protectively immunized with a nonliving vaccine.

Mice protected against Schistosoma mansoni infection by intradermal (i.d.) vaccination with nonliving schistosomula or soluble extracts of larval or adult schistosomes (SCHLAP and SWAP, respectively) produce antibodies that react by Western blot analysis with one antigen of Mr (X 10(-3)) 97 in SWAP prepared in the presence of protease inhibitors and two antigens of Mr (X 10(-3)) 95 and 78 in SWAP prepared in their absence. Vaccine antibodies also immunoprecipitated a single 97k molecule, with a pI of 5.5, from detergent extracts of [35S] methionine-labeled schistosomes. Three hybridomas, produced from spleen cells of i.d. immunized mice, all recognized both the 95k/78k doublet and the 97k antigen, indicating that the two lower Mr components are degradation products of the same 97k molecule. The 97k/95k/78k complex (Sm-97) was purified by affinity chromatography and found to constitute 0.5% of the total protein in SWAP. 125I-concanavalin A bound weakly to purified Sm-97, indicating that this antigen is minimally glycosylated. By indirect immunofluorescence, Sm-97 was localized to regions just below the tegumental and gut syncitia of adult worms. Mice protected by i.d. vaccination produced high titers (1:10,240) of anti-Sm-97 antibodies, whereas chronically infected mice responded at a much lower level (titer 1:640). In contrast, mice protectively immunized with irradiated cercariae and mice nonprophylactically inoculated by the i.v. route failed to produce detectable anti-Sm-97 antibodies. Competitive radioimmunoassays performed with 125I-labeled monoclonal antibodies and purified antigen defined at least two distinct epitopes on Sm-97. Antibodies from i.d. vaccinated mice recognized both monoclonal antibody-defined epitopes, whereas anti-Sm-97 antibodies in chronic infection sera recognized neither. Finally, purified Sm-97 was shown to elicit delayed-type hypersensitivity in i.d. vaccinated mice, suggesting that this molecule is also capable of evoking cell-mediated responses, a finding consistent with its proposed function as a vaccine immunogen.

Expression of monoclonal antibody-defined epitopes of keratin 19 in human tumours and cultured cells

The monoclonal antibodies BA16 and BA17, reacting specifically with human keratin 19 (40 kD) have been tested by immunohistochemical staining methods for their reaction with a wide range of human tumours and cultured cells. Primary adenocarcinoma and their metastases showed a homogeneously positive reaction with > 95% of the tumour cells staining. Non-epithelial tumours, basaliomas and squamous cell carcinomas were unstained, while benign breast lesions and a thyroid adenoma show a mosaic pattern of stained and unstained (5–40%) cells. These three staining patterns were also seen in cultured cells. Positive homogeneous staining was seen in all breast cancer cell lines examined with the exception of PMC42, which exhibits stem cell characteristics, and which showed the heterogeneous pattern of staining seen in milk cell cultures. Non-epithelial lines and strains, two cell lines from cervical carcinomas and three SV40 transformed breast epithelial lines were unstained. The antibodies BA16 and 17 are potentially useful reagents for distinguishing adenocarcinomas (and their metastases) from non-epithelial tumours and from squamous carcinomas. They may also discriminate between benign and malignant breast lesions, and identify a specific differentiation phenotype in the secretory cell lineage.

Location of immunodominant antigenic determinants on fragments of the tick-borne encephalitis virus glycoprotein: Evidence for two different mechanisms by which antibodies mediate neutralization and hemagglutination inhibition

A model showing the topological distribution, functions, and serological specifities of eight distinct, monoclonal antibody-defined epitopes on the tick-borne encephalitis (TBE) virus glycoprotein has been presented in a previous publication (F. X. Heinz, R. Berger, W. Tuma, and Ch. Kunz (1983). Virology 126, 525–537.) In the present report the influence of conformational change, chemical modification, and fragmentation on the antigenic reactivity of each epitope has been analyzed by the use of blocking enzyme immunoassays and “Western blotting.” One of the two major antigenic domains (A), composed of three different epitopes, completely lost its antigenicity upon incubation at pH 5.0 or by treatment with guanidine-HCl/urea, SDS, reduction and carboxymethylation, as well as by proteolytic (trypsin, α-chymotrypsin, thermolysin) and chemical (CNBr) fragmentation. The second major antigenic domain (B), however, defined by four distinct monoclonal antibodies, three of which are hemagglutination (HA)-inhibiting, neutralizing, and protective, was shown to be resistant to low pH, guanidine-HCl/urea treatment, and proteolytic cleavage of the native protein. Also, polyclonal immune sera from mice and rabbits contained antibody populations reactive with antigenic determinants which are resistant and others which are sensitive to conformational change and fragmentation. Glycoprotein fragments with molecular weights of about 9000, generated by proteolysis of the native protein, were immunoreactive with neutralizing and protective monoclonal antibodies (defining domain B) as well as with a polyclonal mouse immune serum. Thus, these fragments appear to contain antigenic determinants which are immunodominant on the native protein and play an important role in the induction of a protective immune response against TBE virus. In addition, these results show that antibody binding to antigenic domains which are topologically and structurally completely unrelated may result in neutralization and/or HA inhibition. As the presence of two receptor-binding sites is unlikely, different effector mechanisms may account for the effects of these antibodies. The antigenic reactivity of domain A is sensitive to the same treatments which also inactivate HA activity of TBE virus, whereas domain B is resistant. These treatments include a change of domain A induced by incubation at slightly acidic pH which also results in inactivation of virus infectivity. Antibodies to domain A therefore presumably block viral activities by direct binding at or near the putative receptor-binding site whereas antibodies to domain B may cause loss of biological activities by inducing a conformational change of the receptor-binding site.

Monoclonal antibody (Tü48) defining alloantigenic class I determinants specific for HLA-Bw4 and HLA-Aw23,-Aw24 as well as -Aw32

A monoclonal antibody (Tii48) was prepared against human lymphocytes. Immunochemical analysis indicated that Tii48 binds to a fraction of HLA molecules. Tii48 was tested on 398 HLA-A,B,C/DR-typed normal blood donors in the microcytotoxicity assay and was found to detect a “supertypic” determinant on molecules bearing the HLA specificities -Bu4. Au23(9). -Au24(9), and -Au32. Among the HLA-Bu4-positive individuals, negative or weak reactivity of Tii48 was found with about one-fourth of normal donors heterozygous for the HLA antigen Bw44. This points to the possibility that the antigenic determinant detected by Tii48 is not expressed on all HLA-molecules carring the HLA-Bu4 specificity on the cell surface. Alternatively, weak or no expression of the Tii48 antigenic determinant on some HLA-Bu44 bearing molecules might be explained by the existence of molecular variants of glycoproteins with this HLA specificity. The concept of “supertypic” HLA specificities is discussed with regard to the expression of a monoclonal antibody-defined epitope on both certain HLA-A and B molecules.