Carbohydrate-associated immunodominant epitope(s) of CA215

RP215 monoclonal antibody (Mab) was initially generated against OC-3-VGH ovarian cancer cells and was shown to react with a cancer-associated carbohydrate epitope in glycoproteins designated as CA215. Additional five high affinity Mabs, designated as RCA-10, -100, -104, -110 and -111, respectively, were generated by using affinity-purified CA215 as the immunogen in this study. All RCA Mabs were found to recognize periodate-sensitive carbohydrate-associated epitope(s) and to pair with RP215 in typical sandwich enzyme immunoassays for the quantification of CA215. When compared with those of RP215, the amino acid sequence homology of the Fab regions ranged from 100% for RCA-100 to 65% for RCA-110, based on which 3 distinct Mab groups were categorized. In vitro TUNEL apoptosis and complement-dependent cytotoxicity assays were performed with these Mabs and found to have comparable inhibitory efficacy to cancer cells. Results of biochemical and immunological assays revealed that RP215, RCA-100 and RCA-10 react with the linear carbohydrate-associated epitope, whereas the others recognize the conformational form of the epitope in CA215. This study has suggested that the unique carbohydrate-associated epitope(s) is immunodominant in mice when immunized with CA215. It remains to be demonstrated if the differential anti-cancer efficacy exists among the distinct groups of these anti-CA215 Mabs.     

Molecular and immuno-characteristics of immunoglobulin-like glycoproteins in cancer cell-expressed biomarker, CA215

RP215 monoclonal antibody (Mab) was shown to recognize a specific carbohydrate-associated epitope found in cancer cell-expressed glycoproteins, known as CA215. The membrane-bound and soluble forms of CA215 were detected in almost all of the cancer cells in humans, but rarely found in normal tissues. Through MALDI-TOF MS analysis, it has been reported previously that as much as 40% of the detected tryptic peptides of CA215 showed high degrees of sequence homology to those found in immunoglobulin heavy chains. The cancer cell-derived immunoglobulins were further purified from CA215 by affinity column-linked with goat anti-human IgG for molecular characterizations. Semi-quantitative RT-PCR was used to determine the mRNA levels of various immunoglobulin genes expressed by cancer cells of single or multi-cell origins and compared with those found in normal human serum. The stability of CA215 was investigated under different experimental conditions. It was observed that the RP215-specific epitope in CA215 is stable at neutral pH, in human serum or in mice (half life of 5-18 days), but unstable at extreme pH's (pH ≤ 2.0; pH ≥ 12.0) or high temperatures. Enzyme immunoassays were performed with several secondary antibody probes related to human IgG. It was demonstrated that cancer cell-expressed immunoglobulins with RP215-specific epitope have much lower immunoactivity than that of normal human IgG (≤ 5%), despite the fact that both showed almost identical amino acid sequence in the respective Fc region reported previously. This could be the result of aberrant glycosylation of CA215 in cancer cells. Aberrant glycosylation of glycoproteins may have important biological implications on the proliferation of cancer cells in vitro or in vivo.     

Molecular and Immuno-Characteristics of Immunoglobulin-like Glycoproteins in Cancer Cell-expressed Biomarker,CA215

RP215 monoclonal antibody (Mab) was shown to recognize a specific carbohydrate-associated epitope found in cancer cell-expressed glycoproteins, known as CA215. The membrane-bound and soluble forms of CA215 were detected in almost all of the cancer cells in humans, but rarely found in normal tissues. Through MALDI-TOF MS analysis, it has been reported previously that as much as 40% of the detected tryptic peptides of CA215 showed high degrees of sequence homology to those found in immunoglobulin heavy chains. The cancer cell-derived immunoglobulins were further purified from CA215 by affinity column-linked with goat anti-human IgG for molecular characterizations. Semi-quantitative RT-PCR was used to determine the mRNA levels of various immunoglobulin genes expressed by cancer cells of single or multi-cell origins and compared with those found in normal human serum. The stability of CA215 was investigated under different experimental conditions. It was observed that the RP215-specific epitope in CA215 is stable at neutral pH, in human serum or in mice (half life of 5–18 days), but unstable at extreme pH’s (pH ≤ 2.0; pH ≥ 12.0) or high temperatures. Enzyme immunoassays were performed with several secondary antibody probes related to human IgG. It was demonstrated that cancer cell-expressed immunoglobulins with RP215-specific epitope have much lower immunoactivity than that of normal human IgG (≤ 5%), despite the fact that both showed almost identical amino acid sequence in the respective Fc region reported previously. This could be the result of aberrant glycosylation of CA215 in cancer cells. Aberrant glycosylation of glycoproteins may have important biological implications on the proliferation of cancer cells in vitro or in vivo.     

Epitope mapping of the HIV-1 gag region with monoclonal antibodies.

A new type of immunochemical mapping of the human immunodeficiency virus type 1 (HIV-1) gag region was performed. By use of native HIV-1 viral lysates or the gag recombinant p24-15 antigen, a new set of monoclonal antibodies (Mabs) to the gag region proteins was generated. Synthetic HIV-1 peptides covering the entire gag region were used to specifically localize the continuous epitopes by direct binding to the Mabs and by blocking the Mab immunoreactivity. The identified immunogenic epitopes were localized between the gag amino acids (aa) 108-127, 203-217, 208-222, 248-282, 273-302, 288-307, 308-322, 331-354 and 408-422. These continuous epitopes formed seven immunogenic regions. One strongly p17-reactive Mab appeared to react with a discontinuous epitope, the components of which were 110 aa distant in the linear sequence: aa 23-27 and 128-132. The synthetic peptides appeared to be more congruent with the Mab-reactive sites in solution than when coated to a solid phase.     

Characterization of conformation-specific monoclonal antibodies against rabies virus nucleoprotein

Three anti-rabies virus (RABV) nucleoprotein (N) monoclonal antibodies (Mab) were characterized by immunofluorescence assays, western blotting, and immunohistochemistry. One of these Mabs recognized the antigen by all of the assays, while the other two recognized N only in the native form in the immunofluorescence assay. These data, together with epitope mapping studies, suggest that two anti-N Mabs recognize conformational epitopes located within the N-terminal region of the RABV N protein. The availability of Mabs specific for both linear and epitope-specific antibodies should prove valuable for rabies diagnosis as well as for RABV N protein structure–function studies.     

Epitope analysis with monoclonal antibodies directed against class II molecules subdivides HLA-DR2, DR3 and DR4: correlations with cellularly defined Dw specificities

Monoclonal antibodies (Mabs) defining 14 distinct polymorphic epitopes have been produced against the class II antigens of HLA-DR3Dw3DQw2 cells. Population analysis indicates that Mab C1 is directed against the DQw2 specificity and Mab M6 against the DRw52 specificity. The remaining Mabs define epitopes shared by the class II molecules of DR3 and various other specificities. Seven DR3Dw3DQw2 haplotypes were examined and could be divided into two types based on the presence of the epitope defined by Mab M3. Analysis of DR2 and DR4 homozygous cells with these Mabs revealed several distinct patterns of epitope expression. These subdivisions were found to correlate with the cellularly defined Dw specificities.     

Identification and characterization of monoclonal antibodies against the ORFV059 protein encoded by Orf virus

Recent outbreaks of orf in China have been attributed to a novel strain of Orf virus (ORFV) designated ORFV-Jilin. Currently, monoclonal antibodies (Mabs) have not yet been developed against this specific pathogen even though such entities could have potential applications regarding the diagnosis and characterization of ORFV-Jilin. Therefore, the current study was undertaken to generate Mab against the immunodominant ORFV059 protein of this virus. For this purpose, the ORFV-Jilin ORFV059 protein was expressed in Escherichia coli and subsequently used as an antigen to immunize mice and for the initial screening of hybridomas prepared from the mice for their ability to produce anti-ORFV059 protein Mabs via an indirect ELISA. Ten, positive hybridomas were identified in this manner and verified based on the ability of their released Mab to react specifically with both naturally and artificially expressed ORFV059 protein in Western blots. The two hybridomas with the greatest propensity to secrete Mab were subcloned three times before being introduced intraperitoneally into mice. Afterwards, both Mab were separately purified from the mice’s ascetic fluids and found to successfully recognize the ORFV-Jilin ORFV059 protein in a variety of immunological assays. Thus, the widespread utility of these Mab as a diagnostic core reagent should prove invaluable for further investigations regarding the mechanisms of orf pathogenesis and the control of this disease. In this regard, it should be noted that Mab A3 was used to confirm the predicted late expression of the ORFV-Jilin ORFV059 protein during virus replication.     

Variable expression of epitopes on the surface of Mycoplastna gattisepticum demonstrated with monoclonal antibodies

Twelve monoclonal antibodies (Mabs) against Mycoplasma gallisepticum (Mg) strains F, R, S6(208) and PET2 were used for analysis of epitopes of 22 Mg strains. Six Mabs recognized surface epitopes in the majority of strains, but did not react with variant strains like K 503 and K 703. Two Mabs reacted with epitopes on about 56 kilodalton (kDa) proteins and showing consistent expression on Mg colonies. Three Mabs recognized three different variable surface epitopes associated with about 67 kDa proteins and one Mab variable epitope on about 33 and 80 kDa proteins. Two-dimensional immunoblotting showed considerable differences in the charge of proteins bearing variable surface epitopes in different Mg strains. Subcloning of four low passage Mg strains using Mabs for screening populations that derived from a single colony with defined surface epitopes showed that some colonies may switch surface epitopes associated with 67 and 80 kDa proteins. This switching was reversible and generated subpopulations of Mg expressing different combinations of surface epitopes. Phenotypic switching of epitopes probably occurs also in vivo and may be the mechanism enabling Mg to evade the host immune response.     

Use of monoclonal antibodies for the identification of different antigenic domains in apple chlorotic leaf spot virus

Summary Thirteen monoclonal antibodies (Mabs) specific for apple chlorotic leaf spot virus (ACLSV), produced by the somatic cell hybridization technique, were used to investigate the antigenic structure of the virus. Epitope specificity studies showed that these Mabs defined in ACLSV particles seven independent antigenic domains, representing at least eight distinct epitopes. One of them was present only in virions and not in dissociated subunits. It appeared that the interaction between a Mab and the virus could, in some cases, induce conformational changes in the viral particles which enhanced the binding of others. Twenty nine virus isolates differing in geographical origin, primary hosts and symptomatology were tested with these monoclonal antibodies by ELISA. With the exception of two Mabs which did not react with three cherry isolates, and one Mab which did not react with one plum isolate, all of them recognized all ACLSV isolates tested.     

Monoclonal antibodies to the haemagglutinin HA1 subunit of the pandemic influenza A/H1N1 2009 virus and potential application to serodiagnosis

In order to provide specific serological reagents for pandemic influenza A/H1N1 2009 virus, monoclonal antibodies (Mabs) to recombinant haemagglutinin component HA1 (rHA1) were generated after fusing spleen cells from a mouse immunized with rHA1 protein derived from influenza strain A/California/06/09 H1N1 with a mouse myeloma cell line. Five hybridoma clones secreting Mabs specific for the rHA1 protein derived from pandemic influenza A/H1N1 2009 and not for rHA1 from seasonal H1N1 influenza strains A/Brisbane/59/07 and A/Solomon Islands/03/06 were identified by EIA. Mabs 7H4, 9A4, and 9E12 were reactive in Western blots with full length rHA and/or rHA1 subunit derived from A/California/06/09 strain. Only Mab 1F5 inhibited haemagglutination of turkey red blood cells with recombinant NIBRG‐121 virus derived from A/California/07/09, but did not react in Western blots. Immunostaining of MDCK cells infected with NIBRG‐121 was localized to the membrane/cytoplasm for four of the reactive Mabs. The differing reactivity of the Mabs in Western blots, immunostaining, EIA, and haemagglutination inhibition assay suggest that at least four of the five Mabs recognize different epitopes on HA1 of the pandemic influenza A/H1N1 2009 virus. Ferret antisera to pandemic influenza A/H1N1 2009 (A/England/195/09 and A/California/07/09 strains) and sera from human subjects vaccinated with Influenza A (H1N1) 2009 Monovalent Vaccine (CELTURA®, Novartis Vaccines, Germany), inhibit binding of 1F5‐HRP to biotinylated rHA1 derived from A/California/06/09 in a competitive EIA, suggesting that the epitope recognized by this Mab also evokes an antibody response in infected ferrets and vaccinated humans. J. Med. Virol. 83:559–567, 2011. © 2011 Wiley‐Liss, Inc.     

Characterisation of novel linear antigen epitopes on North American-type porcine reproductive and respiratory syndrome virus M protein

The M protein, encoded by the porcine reproductive and respiratory syndrome virus (PRRSV) ORF6 gene, is considered to be one of the most conserved PRRSV proteins. In recent decades, highly specific monoclonal antibodies (Mabs) have been exploited to provide reliable diagnoses for many diseases. In this study, two different Mab clones targeting the linear epitopes on the PRRSV M protein were generated and characterized. Both Mabs showed binding activity against the native PRRSV virion and recombinant M protein when analyzed by immunofluorescence assay (IFA) and Western blot. The targeted epitope of each Mab was mapped by serial truncation of the M protein to generate overlapping fragments. Fine epitope mapping was then performed using a panel of expressed polypeptides. The polypeptide sequences of the two epitopes recognized by Mabs 1C8 and 3F7 were ³SSLD⁶ and ¹⁵⁵VLGGRKAVK¹⁶³, respectively, with the former being a newly identified epitope on the M protein. In both cases, these two epitopes were finely mapped for the first time. Alignments of Mab epitope sequences revealed that the two epitopes on the M protein were highly conserved between the North American-type strains. These Mabs, along with their mapped epitopes, are useful for the development of diagnostic and research tools, including immunofluorescence, ELISA and Western blot.     

Monoclonal antibodies to recombinant human laminin-binding protein. Production and immunochemical description

Thirteen murine hybridoma lines producing monoclonal antibodies (Mabs) to recombinant human laminin-binding protein (rLBP) were developed. All 13 Mabs reacted with affinity purified 43 kDA rLBP in ELISA and Western blotting. Mab class determination showed 9 Mabs as belonging to IgM class, 2 Mabs--to IgG2 subclass, 1 Mab--to IgG1 and 1 Mab--to IgG2b. Ten Mabs of different classes were capable to react with LBP on the surface of Vero cells. Mabs displayed a high and simultaneously varying affinity to rLBP (10(8) 10(9) M(-1)). The Mab affinity was found to be comparable with the mean affinity of mouse and rabbit antibodies isolated from hyperimmune sera. The possibility of using the produced Mabs in mapping the LBP domains involved in virus attachment, cell differentiation and cancer metastases progression as well as in the systemic response to bacterial protozoan and parasitic infection is under discussion.     

Monoclonal antibodies against human IgE. Identification of a determinant restricted to IgE of the lambda light-chain type

The specificity of five monoclonal anti-IgE antibodies (Mabs) was studied in direct latex agglutination and agglutination-inhibition experiments by particle-counting immunoassay. Twenty IgE myeloma proteins and several purified D epsilon O-, D epsilon 2-containing pepsin and papain fragments of IgE-DES(kappa) were used in the evaluation. The results demonstrate two Mabs with isotypic specificity for two distinct epitopes of the Fc epsilon-fragment within the D epsilon 1- and D epsilon 2-determinants. One Mab recognized only the immunizing IgE protein and was directed against determinants on the Fd epsilon-fragment probably related to the idiotype. Anti-Em(1) allotypic Mabs recognized all 20 IgE myeloma proteins including two of Japanese origin and the Em(1)-allotype was confined to D epsilon-determinants. Interestingly, one Mab (ALE) reacted with all 8 IgE myeloma proteins of the lambda light-chain type but none out of 12 bearing kappa chains. ALE seems therefore to recognize a new marker on IgE besides the known idiotypic, allotypic and isotypic ones. These results illustrate that a critical specificity control of Mabs is always warranted. Moreover, one should be aware of possible interference in IgE assays from the kind of determinants recognized by ALE whenever intact IgE myeloma proteins are used to raise polyclonal antisera, to get immunosorbent-purified anti-IgE antibodies or when used as tracers and standards.     

Monoclonal antibodies against avian paramyxovirus-3: antigenic mapping and functional analysis of the haemagglutinin-neuraminidase protein and the characterization of nonspecific monoclonal antibodies

Summary Sixteen monoclonal antibodies (Mabs) which immunoprecipitated the haemagglutinin neuraminidase (HN) of chorio-allantoic membrane-grown avian paramyxovirus-3 (PMV-3) of British turkeys were produced. Thirteen were PMV-3 specific. Three were nonspecific because they also bound to other viral proteins and to bovine kidney cells treated with neuraminidase enzyme or infected with influenza virus.The thirteen specific Mab defined four antigenic regions A-D by competition and variant selection assays. Region A was subdivided into five epitopes and region B into two epitopes. The thirteen Mab neutralized and were active in haemagglutination inhibition (HI) and twelve were active in haemolysis inhibition (HLI) tests. Neuraminidase inhibition (NI) was epitope-dependent.Mabs to five of the epitopes A₁, A₂, A₃, A₅, and C bound to the 1981 British turkey isolates but not the 1968 American turkey isolate. The Mab to epitope A₄ bound to both viruses. The Mabs to epitopes B₁, B₂, and D also bound to a parakeet isolate of PMV-3 which was the third PMV-3 tested. The Mab to B₂ gave identical titres to all three viruses and had HI, HLI, and NI activities. This made it a potential diagnostic reagent for avian PMV-3 viruses.One of the nonspecific Mabs bound to lactose-like moeities as reported on influenza virus and one to maltose-like moeities as on retroviruses. Immunoglobulin from all three nonspecific Mab had some HI activity.     

Partial characterization of an antigenic site of high molecular weight basic antigen, a ryegrass pollen allergen, using a monoclonal antibody

We have previously reported on the production of murine monoclonal antibodies (Mabs) to the retentate fraction of the aqueous extract of Kentucky bluegrass pollen (KBG-R) [Kisil et al. (1980) Fedn Proc. 39, 3479]. In the present study, the ability of one of these Mabs (Mab 12) to recognize various antigenic components present in KBG-R and the corresponding fraction of ryegrass (rye-R) was evaluated by the Western and immunoblot methods. Thus, KBG-R and rye-R were resolved electrophoretically into a large number of components. Remarkably, the concurrent immunoblot analysis with Mab 12 detected only a single antigenic component in each of the retentate fractions. The position of the antigenic component observed on these immunoblots was identical to that obtained with the rye allergen high mol. wt basic antigen (mol. wt 56,800). To characterize the antigenic site recognized by the Mab, the size of HMBA was reduced by cleavage with CNBr, the resulting fragments separated by high-performance liquid chromatography on a reverse-phase column and their antigenic activity analyzed by immunoblot. Two peptides, CB-1 (mol. wt = 17,400) and CB-2 (mol. wt = 22,000), retained the capacity to react with Mab 12 and also IgE antibodies present in a pool of sera from grass allergic individuals.     

Production and characterization of monoclonal antibodies to Ha-ras and N-ras p21

The mammalian ras family consists of the Ha, Ki and N-ras genes that encode a series of 21,000 dalton proteins (p21). The three ras proteins participate in normal cell physiology and have been implicated in cellular transformation by either overexpression of the normal p21 or by mutation at positions 12, 13, or 61. To help understand the biological roles of the different ras proteins, we have generated monoclonal antibodies (Mabs) to the Ha-ras and N-ras p21. Mab Ha-770, raised to a Ha-specific synthetic peptide, reacts with Ha-ras recombinant p21 (r-p21) as well as cellular Ha-ras p21 by immunoprecipitation. Western blot and sandwich ELISA assays. Mab N-838, raised to an N-ras specific synthetic peptide, reacts with the N-ras recombinant p21 by immunoprecipitation, Western blot and sandwich ELISA assays. Mabs to the Ha-ras and N-ras p21 should be valuable reagents in assessing the individual roles of ras proteins in normal and neoplastic cells.     

Detection of specific forms of plasminogen activator inhibitor type 1-by monoclonal antibodies

Monoclonal antibodies to plasminogen activator inhibitor type-1 (PAI-1) were generated and characterised for their ability to inhibit PAI-1 interaction with tissue plasminogen activator (t-PA) and urokinase (u-PA) and detection of the various forms of PAI-1 (native, complexed, and degraded) by immunoblotting. Mab17 inhibited both complex formation between PAI-1 and t-PA/u-PA and PAI-1 activity in a dose dependent manner by 90%. Mab 25 was much less effective, blocking complex formation less than 30% and PAI-1 activity less than 20%. The Kds of Mab 17 and Mab25 were 2.8×10⁻¹¹ M and 2.6×10⁻¹⁰ M, respectively. Following SDS-PAGE and immunoblotting, Mab 17 detected native PAI-1 only; PAI-1 in complex and the t-PA/u-PA degraded form of PAI-1(M_r=42000) did not react with this antibody. In contrast, Mab25 detected all three forms of PAI-I although the affinity for the native form appeared to be greater than Mab 17 or the PAI-1 polyclonal employed. Despite these differences, both monoclonal antibodies immunoprecipitated native and degraded PAI-1 equally as well. These results suggest that the epitope of Mab 17 is associated with the reactive site of PAI-1 and that this region is either missing or not accessible in the cleaved form or in complex.     

Epitope analysis of capsid and matrix proteins of North American ovine lentivirus field isolates.

Monoclonal antibodies (MAbs) directed against two phenotypically distinct ovine lentivirus (OvLV) strains were generated by fusion of BALB/c SP2/0-Ag 14 myeloma cells with spleen cells from mice immunized with purified OvLV. Hybridomas were selected by indirect enzyme-linked immunosorbent assay (ELISA) and analysis of reactivity on immunoblots. The majority (17 of 21) of the MAbs recognized the gag-encoded capsid protein, CA p27, of both strains. Four other MAbs recognized a smaller structural protein, presumably a matrix protein, MA p17. Three distinct epitopes on CA p27 and one on MA p17 were distinguished by the MAbs with competition ELISA. MAbs from each epitope group were able to recognize 17 North American field isolates of OvLV and the closely related caprine arthritis-encephalitis virus (CAEV). Analysis of the data indicated that these epitopes were highly conserved among naturally occurring isolates. A representative MAb from each epitope group of anti-CA p27 MAbs reacted with four field strains of OvLV and CAEV on immunoblots. An anti-MA p17 MAb recognized the same OvLV strains on immunoblots but failed to recognize CAEV. MAbs which recognize conserved epitopes of gag-encoded lentivirus proteins (CA p27 and MA p17) are valuable tools. These MAbs can be used to develop sensitive diagnostic assays and to study the pathogenesis of lentivirus infections in sheep and goats.     

Quantitative ELISA for human lactate dehydrogenase isoenzyme 5

Four monoclonal antibodies (Mab) derived from mice immunized with lactate dehydrogenase 5 (LDH5) react strongly with LDH5, but weakly with LDH2 which contains a single subunit of type M. Experimental evidence suggest that these antibodies are directed to an antigenic determinant in the interface between two subunits of type M. A sandwich ELISA procedure was devised, using these Mabs to identify and quantify LDH5. The procedure involves immobilization of one of these Mabs by its adsorption onto polyclonal anti-mouse IgG coated polystyrene plates, adsorption of LDH5, its identification by the same Mab as that used in the immobilization step, and finally color development by an enzyme labeled rabbit anti-mouse IgG antiserum. The method enables LDH5 to be assayed at a concentration range of 0-5 micrograms/ml.     

A two-site monoclonal antibody ELISA for the quantification of the major Dermatophagoides spp. allergens, Der p I and Der f I

A two-site monoclonal antibody (Mab) ELISA was developed to measure the Group I allergens from Dermatophagoides spp., Der p I from D. pteronyssinus and Der f I from D. farinae. Species-specific Mabs were used to coat microtiter plates which were then incubated with allergen or house dust extracts. Bound allergen was detected using a biotinylated Mab which recognized a common epitope on both Der p I and Der f I, followed by the addition of streptavidin-peroxidase and ABTS/H2O2 substrate. The assay had low non-specific binding (approximately 0.08 absorbance units) and had a sensitivity of 5 ng/nl for aqueous allergen extracts (equivalent to 0.1 microgram allergen/g dust). 53 dust samples were assayed using the Mab ELISA and an RIA previously described using 125I-labelled Mab. The results showed a very good quantitative correlation between the assays (r = 0.96, p less than 0.001 for Der p I; r = 0.92, P less than 0.001 for Der f I). A further 132 dust samples from a different geographical areas were also assayed by both methods and gave correlation coefficients of 0.90 (P less than 0.001) and 0.86 (P less than 0.001) for Der p I and Der f I, respectively. The Mab ELISA will be useful in epidemiological studies of allergic asthma, both in the assessment of levels of dust mite allergen present in houses and the efficacy of allergen avoidance regimes.