Development of monoclonal antibodies against bovine mucin core 2 β6 N-acetylglucosaminyltransferase

Molecular cloning techniques have been used to produce abundant amounts of recombinant glycosyltransferases for biochemical studies. We recently cloned a cDNA which encoded bovine mucin core 2 6N-acetylglucosaminyl transferase (C2TF). Poly-histidine-C2TF fusion protein was generated from the cloned cDNA in the E. coli Xpress system and used to produce monoclonal antibodies (MAbs). We obtained seven hybridomas which secreted MAbs against bovine C2TF in mouse ascites with titers ranging from 1:1280 to 1:40960 as assessed by immunofluorescence assay (IF). Isotyping revealed that all seven MAbs were IgG (4 IgG1, 2 IgG2b and 1 IgG2a). The affinity constants (M^–2) for these MAbs range from 5.4 × 10⁷ to 1.2 × 10⁹. These MAbs recognized bovine C2TF in tissue sections and on Western blottings. Six of these MAbs reacted with human core 2-M enzyme and one with both core 2-L and core 2-M enzymes on Western blottings. Therefore, These antibodies should be useful for further study of bovine and human core 2 enzymes.     

Altered reactivity of immunoglobulin produced by human-human hybridoma cells transfected by pSV2-neo gene

The HB4C5 and HF10B4 cell lines are human-human hybridomas producing human IgM monoclonal antibodies (MAbs) reactive to porcine carboxypeptidase A (CPase), but not to double stranded DNA (ds DNA). We obtained G418-resistant HB4C5 and HF10B4 cells by an introduction of pSV2-neo DNA. Almost all of the G418-resistant clones produced MAbs reactive to not only the CPase but the ds DNA. The results of the inhibition ELISA suggested that the cross-reactivity of the antibodies from G418-resistant clones to CPase and ds DNA was responsible for the alteration on their antigen specificity. HB4C5 and HF10B4 cells and their G418-resistant clones produced antibodies having glycosylated chain. The antibodies produced by tunicamycin-treated G418-resistant subclones of HB4C5 and HF10B4 lost the ability to bind to ds DNA, but retained the ability to bind to CPase. These results suggest that an introduction of pSV2-neo DNA into these hybridomas alters the specificities of their MAbs, and that the alteration to antigen binding specificities of their MAbs may be associated with glycosylation of the MAbs by these hybridomas.     

The molecular nature of Fucus serratus sperm surface antigens recognised by monoclonal antibodies FS1 to FS12

Sperm of the brown alga Fucus serratus are highly differentiated, biflagellate, naked cells. Immunolocalisation studies, employing monoclonal antibodies (MAbs — designated FS1 to FS12) raised against antigens of these sperm cells, have revealed that some sperm surface components are distributed over the entire cell, whereas others are restricted to, or occur preferentially on, the surface of the anterior flagellum or cell body. This report describes the use of these MAbs in Western-blot procedures and antigen-modification binding assays to determine the nature of these sperm surface components. Monoclonal antibodies which bind to antigens found on the cell body and both flagella (FS3, FS4, FS6, FS8, FS10) recognise carbohydrate epitopes of a high-molecular-weight glycoprotein (M_r=205 kDa). These MAbs were initially chosen at random from a much larger number of antibodies which bound to sperm in a similar fashion, indicating that this glycoprotein is an immunodominant antigen. Though these MAbs compete under conditions of limited antigen availability, differences in the effects of periodate on antibody binding and differences in other binding data indicate that the MAbs recognise epitopes of this glycoprotein which are neighbouring or overlapping, rather than common. The MAb FS9, which has a similar binding pattern to the above antibodies, also seems to bind to carbohydrate epitopes, but the antigen recognised by this antibody could not be identified in Western-blotting procedures. The MAbs FS7 and FS12, which bind to the mastigonemes on the anterior flagellum and to the cell body and posterior flagellum, recognise a set of glycoproteins in the molecular-weight range 40–250 kDa. The evidence indicates that the antibodies are binding to N-linked carbohydrate side chains of these glycoproteins. Three MAbs that bind to the anterior flagellum (FS2, FS5 and FS11) recognise protein antigens in the molecular-weight range 90–250 kDa; it is not known whether these antigens are glycosylated. The MAb FS1, which binds primarily to the sperm cell body, could not be used in enzyme-linked immunosorbent assays or Western-blotting procedures and the antigen recognised by this antibody is so far uncharacterised.Abbreviations ELISA enzyme linked immunosorbent assay - HRP-RAMIG horseradish-peroxidase-labelled rabbit anti mouse immunoglobulin - Ig immunoglobulin - kDa kilodalton - MAb monoclonal antibody - M_r relative molecular mass - PBS phosphate-buffered saline - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis We are grateful to AFRC for financial support under the cell signalling initiative.     

Inhibition of human immunodeficiency virus type 1 gp120 presentation to CD4 T cells by antibodies specific for the CD4 binding domain of gp120

Human immunodeficiency virus (HIV)-specific CD4 T-cell responses, particularly to the envelope glycoproteins of the virus, are weak or absent in most HIV-infected patients. Although these poor responses can be attributed simply to the destruction of the specific CD4 T cells by the virus, other factors also appear to contribute to the suppression of these virus-specific responses. We previously showed that human monoclonal antibodies (MAbs) specific for the CD4 binding domain of gp120 (gp120(CD4BD)), when complexed with gp120, inhibited the proliferative responses of gp120-specific CD4 T-cells. MAbs to other gp120 epitopes did not exhibit this activity. The present study investigated the inhibitory mechanisms of the anti-gp120(CD4BD) MAbs. The anti-gp120(CD4BD) MAbs complexed with gp120 suppressed gamma interferon production as well as proliferation of gp120-specific CD4 T cells. Notably, the T-cell responses to gp120 were inhibited only when the MAbs were added to antigen-presenting cells (APCs) during antigen pulse; the addition of the MAbs after pulsing caused no inhibition. However, the anti-gp120(CD4BD) MAbs by themselves, or as MAb/gp120 complexes, did not affect the presentation of gp120-derived peptides by the APCs to T cells. These MAb/gp120 complexes also did not inhibit the ability of APCs to process and present unrelated antigens. To test whether the suppressive effect of anti-gp120(CD4BD) antibodies is caused by the antibodies' ability to block gp120-CD4 interaction, APCs were treated during antigen pulse with anti-CD4 MAbs. These treated APCs remained capable of presenting gp120 to the T cells. These results suggest that anti-gp120(CD4BD) Abs inhibit gp120 presentation by altering the uptake and/or processing of gp120 by the APCs but their inhibitory activity is not due to blocking of gp120 attachment to CD4 on the surface of APCs.     

Antigenic analysis of equine infectious anemia virus (EIAV) variants by using monoclonal antibodies: epitopes of glycoprotein gp90 of EIAV stimulate neutralizing antibodies.

Monoclonal antibodies produced against the prototype cell-adapted Wyoming strain of equine infectious anemia virus (EIAV), a lentivirus, were studied for reactivity with the homologous prototype and 16 heterologous isolates. Eighteen hybridomas producing monoclonal antibodies (MAbs) were isolated. Western blot (immunoblot) analyses indicated that 10 were specific for the major envelope glycoprotein (gp90) and 8 for the transmembrane glycoprotein (gp45). Four MAbs specific to epitopes of gp90 neutralized prototype EIAV infectivity. These neutralizing MAbs apparently reacted with variable regions of the envelope gp90, as evidenced by their unique reactivity with the panel of isolates, suggesting recognition of at least three different neutralization epitopes. The conformation of these epitopes appears to be continuous, as they resisted treatment with sodium dodecyl sulfate and reducing reagents. Monoclonal antibodies that reacted with conserved epitopes on gp90 or gp45 failed to neutralize EIAV. Our data also demonstrated that there was a large spectrum of possible EIAV serotypes and confirmed that antigenic variation occurs with high frequency in EIAV. Moreover, the data showed that variation is a rapid and random process, as no pattern of variant evolution was evident by comparison of 13 isolates from parallel infections. These results represent the first production of neutralizing MAbs specific for a lentivirus glycoprotein and document alterations in one or more neutralization epitopes of the major surface glycoprotein among sequential isolates of EIAV recovered during persistent infection.     

Enhancement of antibody production by the addition of Coenzyme-Q<Subscript>10</Subscript>

Recently, there has been a growing demand for therapeutic monoclonal antibodies (MAbs) on the global market. Because therapeutic MAbs are more expensive than low-molecular-weight drugs, there have been strong demands to lower their production costs. Therefore, efficient methods to minimize the cost of goods are currently active areas of research. We have screened several enhancers of specific MAb production rate (SPR) using a YB2/0 cell line and found that coenzyme-Q₁₀ (CoQ₁₀) is a promising enhancer candidate. CoQ₁₀ is well known as a strong antioxidant in the respiratory chain and is used for healthcare and other applications. Because CoQ₁₀ is negligibly water soluble, most studies are limited by low concentrations. We added CoQ₁₀ to a culture medium as dispersed nanoparticles at several concentrations (Q-Media) and conducted a fed-batch culture. Although the Q-Media had no effect on cumulative viable cell density, it enhanced SPR by 29%. In addition, the Q-Media had no effect on the binding or cytotoxic activity of MAbs. Q-Media also enhanced SPR with CHO and NS0 cell lines by 30%. These observations suggest that CoQ₁₀ serves as a powerful aid in the production of MAbs by enhancing SPR without changing the characteristics of cell growth, or adversely affecting the quality or biological activity of MAbs.     

Tumor localization and radioimaging with mixtures of radioiodinated monoclonal antibodies directed to different colon cancer associated antigens

After demonstrating enhanced tumor cell binding with a mixture of monoclonal antibodies (MAbs) in vitro, biodistribution and immunoscintigraphy studies with 3 radioiodinated anti-colon cancer MAbs and a non-specific control MAb (MOPC) were conducted in a human colon cancer (GW-39)-hamster model system. Each of the specific MAbs, but not MOPC, demonstrated extensive tumor binding and in scintigrams affected visualization of all large tumors (>0.85 g) over background. Using single MAbs, few small tumors (0.19–0.50 g) were defined above background (0–29%). However, with combinations of these specific MAbs small tumors were more frequently defined in scintigrams (43–67%). Radioimages using higher doses of MAbs and small, younger tumors more clearly demonstrated the superiority of a MAb mixture. These results confirmed that combinations of MAbs to different antigens can detect smaller tumors with better tumor localization when compared to component MAbs used singly. This study supports the concept that tumor targeting and detection may be enhanced with appropriate mixtures of MAbs.     

A Specific Method for the Detection of Hiochi Bacteria Using an Enzyme-linked Immunosorbent Assay System with Anti-hiochi Bacteria Monoclonal Antibodies

Ten standard strains of hiochi bacteria were selected based on the SDS-PAGE patterns of their cellular proteins. We then obtained ten hybridoma systems that secreted highly reactive monoclonal antibodies (MAbs) to the whole cells of each strain. It was apparent that these MAbs were highly reactive and specific for hiochi bacterial whole cells when using an enzyme-linked immunosorbent assay (ELISA). Three MAbs (two against the homo-fermentative hiochi lactobacilli and an MAb against the hetero-fermentative true hiochi bacilli) showed cross-reactivity to some of the other strains of lactobacilli tested. However, the other MAbs did not react with strains other than the immunogen. A sensitive ELISA method for the detection of hiochi bacteria was examined. It was possible to detect the order of 10³ cells of the ten standard strains. Using this procedure and a mixture of the ten MAbs, a detection limit of 10⁴ cells or less could be obtained for 98.2% of the hiochi bacteria isolated from sake brewing factories. Thus, this immunological technique using MAbs specific for hiochi bacteria is a sensitive and rapid detection method for hiochi bacteria, which can be used in the quality control of sake.     

Monoclonal Antibodies That Bind to Domain III of Dengue Virus E Glycoprotein Are the Most Efficient Blockers of Virus Adsorption to Vero Cells

The specific mechanisms by which antibodies neutralize flavivirus infectivity are not completely understood. To study these mechanisms in more detail, we analyzed the ability of a well-defined set of anti-dengue (DEN) virus E-glycoprotein-specific monoclonal antibodies (MAbs) to block virus adsorption to Vero cells. In contrast to previous studies, the binding sites of these MAbs were localized to one of three structural domains (I, II, and III) in the E glycoprotein. The results indicate that most MAbs that neutralize virus infectivity do so, at least in part, by the blocking of virus adsorption. However, MAbs specific for domain III were the strongest blockers of virus adsorption. These results extend our understanding of the structure-function relationships in the E glycoprotein of DEN virus and provide the first direct evidence that domain III encodes the primary flavivirus receptor-binding motif.     

Monoclonal Antibodies to Distinct Sites on Herpes Simplex Virus (HSV) Glycoprotein D Block HSV Binding to HVEM

HVEM (for herpesvirus entry mediator) is a member of the tumor necrosis factor receptor superfamily and mediates entry of many strains of herpes simplex virus (HSV) into normally nonpermissive Chinese hamster ovary (CHO) cells. We used sucrose density centrifugation to demonstrate that purified HSV-1 KOS virions bind directly to a soluble, truncated form of HVEM (HVEMt) in the absence of any other cell-associated components. Therefore, HVEM mediates HSV entry by serving as a receptor for the virus. We previously showed that soluble, truncated forms of HSV glycoprotein D (gDt) bind to HVEMt in vitro. Here we show that antibodies specific for gD, but not the other entry glycoproteins gB, gC, or the gH/gL complex, completely block HSV binding to HVEM. Thus, virion gD is the principal mediator of HSV binding to HVEM. To map sites on virion gD which are necessary for its interaction with HVEM, we preincubated virions with gD-specific monoclonal antibodies (MAbs). MAbs that recognize antigenic sites Ib and VII of gD were the only MAbs which blocked the HSV-HVEM interaction. MAbs from these two groups failed to coprecipitate HVEMt in the presence of soluble gDt, whereas the other anti-gD MAbs coprecipitated HVEMt and gDt. Previous mapping data indicated that site VII includes amino acids 11 to 19 and site Ib includes 222 to 252. The current experiments indicate that these sites contain residues important for HSV binding to HVEM. Group Ib and VII MAbs also blocked HSV entry into HVEM-expressing CHO cells. These results suggest that the mechanism of neutralization by these MAbs is via interference with the interaction between gD in the virus and HVEM on the cell. Group Ia and II MAbs failed to block HSV binding to HVEM yet still neutralized HVEM-mediated entry, suggesting that these MAbs block entry at a step other than HVEM binding.     

Optimal construction of non-immune scFv phage display libraries from mouse bone marrow and spleen established to select specific scFvs efficiently binding to antigen

Monoclonal antibodies (MAbs) are widely applied in basic research, medicine, and the pharmaceutical industry. Recently, applications and generations of MAbs have been increasingly attracting attention in many research areas since MAbs could be produced in large quantities with the development of genetic technology and antibody engineering. On the other hand, in recent years, phage display system has been developed for high-throughput isolation and generation of novel MAbs that have high affinity with various antigens. This technology is capable of constructing "Library" containing billions of phage repertoires displaying various antibody fragments, and rapid selection of a specific MAb from this phage library. Additionally, this technology has a great advantage that MAbs can be generated without immunization to animals. However, there are still relatively few reports confirming that useful MAbs can be derived from non-immune antibody libraries. The latter, as undertaken by current methods, seem unable to achieve the high quality required to produce useful MAbs for any desired antigen because cloning of antibody gene from non-immune donors is inefficient. This problem is caused by the fact that their RT-PCR primer sets, PCR conditions, and efficiency of subcloning through construction of antibody gene library cannot encompass all the antibody diversity. In an attempt to overcome some of these earlier problems, here we describe an optimized method to establish a high quality, non-immune library from mouse bone-marrow and spleen, and assess its diversity in terms of content of multiple antibodies for a wide antigenic repertoire. As an example of the application of the methodology, we describe the selection of specific MAbs binding to Luciferase and identify at least 18 different clones. Using this non-immune mouse antibody library, we also obtained MAbs for VEGF, VEGF receptor 2, TNF-alpha, and Pseudomonas Exotoxin, confirming the high quality of the library and its suitability for this application.     

Properties of monoclonal antibodies directed against hepatitis B virus polymerase protein

Hepadnavirus polymerases are multifunctional enzymes that play critical roles during the viral life cycle but have been difficult to study due to a lack of a well-defined panel of monoclonal antibodies (MAbs). We have used recombinant human hepatitis B virus (HBV) polymerase (Pol) expressed in and purified from baculovirus-infected insect cells to generate a panel of six MAbs directed against HBV Pol protein. Such MAbs were subsequently characterized with respect to their isotypes and functions in analytical and preparative assays. Using these MAbs as probes together with various deletion mutants of Pol expressed in insect cells, we mapped the B-cell epitopes of Pol recognized by these MAbs to amino acids (aa) 8 to 20 and 20 to 30 in the terminal protein (TP) region of Pol, to aa 225 to 250 in the spacer region, and to aa 800 to 832 in the RNase H domain. Confocal microscopy and immunocytochemical studies using various Pol-specific MAbs revealed that the protein itself appears to be exclusively localized to the cytoplasm. Finally, MAbs specific for the TP domain, but not MAbs specific for the spacer or RNase H regions of Pol, appeared to inhibit Pol function in the in vitro priming assay, suggesting that antibody-mediated interference with TP may now be assessed in the context of HBV replication.     

Production and characterization of murine monoclonal antibodies against staphylococcal enterotoxins A and E

Six murine monoclonal antibodies (MAbs) against staphylococcal enterotoxin A (SEA) and enterotoxin E (SEE) were prepared by fusion of myeloma cells with mouse spleen cells immunized with SEA and SEE. Of five MAbs to SEA tested, two MAbs were reactive with only SEA, whereas three were specific for both SEA and SEE. On the other hand, one MAb to SEE was found to be specific for only SEE. To study specificities of the combining sites of these MAbs, competitive binding assays with either SEA or SEE and horseradish peroxidase conjugated MAbs were performed using unconjugated MAbs as inhibitors. The results obtained in the assays suggest that different epitopes may be located on SEA and that some of them may be cross-reacting epitopes between SEA and SEE.     

Human monoclonal antibodies to pandemic 1957 H2N2 and pandemic 1968 H3N2 influenza viruses

Investigation of the human antibody response to the 1957 pandemic H2N2 influenza A virus has been largely limited to serologic studies. We generated five influenza virus hemagglutinin (HA)-reactive human monoclonal antibodies (MAbs) by hybridoma technology from the peripheral blood of healthy donors who were born between 1950 and 1968. Two MAbs reacted with the pandemic H2N2 virus, two recognized the pandemic H3N2 virus, and remarkably, one reacted with both the pandemic H2N2 and H3N2 viruses. Each of these five naturally occurring MAbs displayed hemagglutination inhibition activity, suggesting specificity for the globular head domain of influenza virus HA. When incubated with virus, MAbs 8F8, 8M2, and 2G1 each elicited H2N2 escape mutations immediately adjacent to the receptor-binding domain on the HA globular head in embryonated chicken eggs. All H2N2-specific MAbs were able to inhibit a 2006 swine H2N3 influenza virus. MAbs 8M2 and 2G1 shared the V(H)1-69 germ line gene, but these antibodies were otherwise not genetically related. Each antibody was able to protect mice in a lethal H2N2 virus challenge. Thus, even 43 years after circulation of H2N2 viruses, these subjects possessed peripheral blood B cells encoding potent inhibiting antibodies specific for a conserved region on the globular head of the pandemic H2 HA.     

Monoclonal anti-idiotypes induce neutralizing antibodies to enterovirus 70 conformational epitopes.

Monoclonal antibodies (MAbs) directed against the prototype enterovirus 70 (EV-70) strain J670/71 were generated and characterized in order to produce anti-idiotypic MAbs (MAb2s) for use as surrogate immunogens. Western immunoblot and radioimmunoprecipitation assays suggested that all the MAbs recognize conformational epitopes on the virion surface. An EV-70-neutralizing antibody, MAb/ev-12 (MAb1), was selected for the production of MAb2s. Five MAb2s were selected for their capacities to inhibit the interaction of MAb/ev-12 with EV-70 in dot immunobinding inhibition and immunofluorescence assays. In addition, these five MAb2s inhibited virus neutralization mediated by MAb/ev-12, suggesting that they recognize paratope-associated idiotopes. In competition enzyme immunosorbent assays, none of the five MAb2s recognized other neutralizing and nonneutralizing EV-70-specific MAbs, demonstrating that the MAb2s were specific for private idiotopes. Immunization with each of the MAb2s was carried out for the production of anti-anti-idiotypic antibodies (Ab3). All five MAb2s induced an immune response. Moreover, results suggested that they share idiotopes, since MAb2-MAb/ev-12 binding could be inhibited by homologous as well as heterologous Ab3s. Ab3 sera were shown to possess antibodies capable of immunoprecipitating 35S-labeled viral proteins in the same manner as MAb/ev-12. Nine of 15 mice immunized with MAb2s demonstrated Ab3 neutralizing activity specific for the prototype EV-70 strain, J670/71. The potential application of MAb2s to serve as surrogate immunogens for conformational epitopes is substantiated by the results presented in this report.     

A pulmonary influenza virus infection in SCID mice can be cured by treatment with hemagglutinin-specific antibodies that display very low virus-neutralizing activity in vitro.

We have previously shown that a pulmonary influenza virus infection in SCID mice can be cured by treatment with monoclonal antibodies (MAbs) specific for the viral transmembrane protein hemagglutinin (HA) but not for matrix 2. Since both types of MAbs react with infected cells but only the former neutralizes the virus, it appeared that passive MAbs cured by neutralization of progeny virus rather than reaction with infected host cells. To prove this, we selected a set of four HA-specific MAbs, all of the immunoglobulin G2a isotype, which reacted well with native HA expressed on infected cells yet differed greatly (>10,000-fold) in virus neutralization (VN) activity in vitro, apparently because of differences in antibody avidity and accessibility of the respective determinants on the HA of mature virions. Since the VN activities of these MAbs in vitro were differentially enhanced by serum components, we determined their prophylactic activities in vivo and used them as measures of their actual VN activities in vivo. The comparison of therapeutic and prophylactic activities indicated that these MAbs cured the infection to a greater extent by VN activity (which was greatly enhanced in vivo) and to a lesser extent by reaction with infected host cells. Neither complement- nor NK cell-dependent mechanisms were involved in the MAb-mediated virus clearance.     

Establishment of monoclonal antibodies against carbohydrate moiety of gastric mucins distributed in the different sites and layers of rat gastric mucosa

Eight monoclonal antibodies (MAbs), designated RGM21 RGM42, were generated against mucin purified from the rat gastric mucosa. By applying ELISA, all of these MAbs were proved to react not only with the purified mucin, but also with the oligosaccharide mixture obtained from the antigenic mucin by alkaline borohydride treatment. Treatment of the mucin-attached ELISA well with trypsin, sodium periodate or galactose oxidase prior to the addition of the MAb was applied to characterize these MAbs. Histochemical observation indicated that all these MAbs were able to stain the formalin fixed-paraffin embedded sections of the rat gastroduodenal mucosa. Although each of these MAbs reacted with distinct mucus-producing cells localized in particular regions of the gastroduodenal mucosa, their staining specificity could generally be classified into four groups. These MAbs might be useful for estimating the physiological and pathological changes of mucins in the gastric mucosa.     

Development of monoclonal antibodies for simple identification of Vibrio alginolyticus

AIMS: The present study was aimed to produce monoclonal antibodies (MAbs) for simple and specific identification of Vibrio alginolyticus infection in shrimp. METHODS AND RESULTS: Mice were immunized with heat killed V. alginolyticus four times at 2-week intervals. The best response mouse was used for spleen donor in hybridoma production. Screening of hybridoma clones producing desired antibodies was performed by dot blotting against V. alginolyticus and other bacterial species, Western blotting and immunohistochemistry of infected shrimp tissues. Four groups of MAbs were obtained; the first group of MAbs demonstrated their limited specificity only to V. alginolyticus used for immunization, while the second and the third groups recognized all three isolates of V. alginolyticus used for testing. The fourth group of MAbs bound to all three isolates of V. alginolyticus and also recognized Vibrio parahaemolyticus, Vibrio harveyi, Vibrio fluvialis and Vibrio vulnificus but did not bind to Vibrio mimicus, Vibrio cholerae, Vibrio penaeicida and other bacterial species tested. MAbs in groups 1, 2 and 3 were able to use for the detection of bacterial infection in the tissues by means of immunohistochemistry. CONCLUSIONS: MAbs specific to V. alginolyticus was produced. These MAbs can be used for specific identification of the bacteria by simple 'dot blotting' method and immunohistochemistry. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated an immunological tool that can be used for simple and accurate identification of V. alginolyticus as well as for the diagnosis of V. alginolyticus infection in animals. This immunological tool can replace costly and laborious biochemical tests.     

Specific identification of pathogenic Yersinia enterocolitica by monoclonal antibodies generated against recombinant attachment invasion locus (rAil) protein

Classical pathogenic strains of Yersinia enterocolitica produce a 17 kDa outer membrane protein, Ail (attachment-invasion locus), which mediates bacterial attachment to some cultures epithelial cell lines and invasion of others. In the present study, hybridomas were developed for the production of monoclonal antibodies (MAbs) against Ail protein of Y. enterocolitica. A set of five stabilized hybridoma cell lines were generated, of which, two MAbs, YEA 302 and YEA 303, exhibited specific reaction to the native Ail protein (17 kDa) present in whole cell lysate of Y. enterocolitica strains beside having reaction with rAil. The other three MAbs, YEA 5, 17 and 32, had some cross reactions with proteins other than Ail. Two out of five MAbs were IgG1, two were IgG2b and one in IgM in nature. MAbs (YEA 302 and YEA 303) did not show any cross-reaction with whole cell lysate of Brucella abortus, Vibrio cholerae, Salmonella typhimurium and Escherichia coli and other species of Enterobacteriaceae including Y. pestis in ELISA and Western blot analysis. The presence of Ail protein among the strains recovered from pork and milk samples was evaluated by these sets of MAbs and the results were compared with the duplex PCR. Collectively, the data suggest that these MAbs may have the potential for their use in the detection of pathogenic Y. enterocolitica reliably, rapidly and at a relatively low cost.     

Criteria for selecting monoclonal antibodies with respect to accumulation in melanoma tissue

Summary Immunohistology provides a necessary but insufficient criterion for selecting monoclonal antibodies (MAbs) capable of tumour targeting in vivo. Additional selection procedures have been evaluated using a panel of anti-melanoma MAbs, including immunoreactivity of (labelled) MAbs, antibody affinity, kinetics of binding and release, apparent antigen density and accumulation in nude mouse transplants. According to these criteria, MAbs M.2.7.6 and M.2.9.4 showed the most favourable properties, i.e. high immunoreactivity and pronounced internalization into melanoma cells. With MAbs M.2.10.15 and KG 6–56, moderate immunoreactivity and a binding pattern characterized by temperature dependence in the absence of internalization was observed. According to the paired label assay, all four MAbs showed specific accumulation into solid melanoma tissue. However, application in the patient still requires evaluation of the side effects of antigen cross-expression on normal human tissues.