Characterization and heterogeneity of monoclonal antibodies directed to intestinal mucin derived from Crohn's disease small bowel

A panel of 12 monoclonal antibodies were produced by immunization of Balb/c mice with small bowel epithelial cells obtained from a patient with active well-documented Crohn's disease. The clones were derived by screening with immunofluorescence microscopy; those with staining patterns suggestive of mucin directed epitopes were chosen for study. Several distinct patterns of staining reactivity were noted, including reagents with homogeneous, luminal, heterogeneous and peripheral goblet cell activity. In addition, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analysis revealed reactivity to high molecular weight mucin. The reactive antigen was resistant to proteinase digestion. No endoneuraminidase activity was detected; however, one neuraminidase sensitive sialic acid epitope was demonstrated. Confirmation of glycoprotein epitopes was accomplished by testing purified mucins from several areas of the gastrointestinal tract by ELISA. Finally, individual small bowel goblet cell heterogeneity was demonstrated by immunofluorescence, Western blotting, and antibody affinity chromatography. These data demonstrate both by morphology and specific binding of antibody affinity chromatography a significant degree of small bowel goblet cell mucin heterogeneity.     

Characterization of antihuman IFNAR-1 monoclonal antibodies: epitope localization and functional analysis.

The type I interferon receptor (IFNAR) is composed of two subunits, IFNAR-1 and IFNAR-2, encoding transmembrane polypeptides. IFNAR-2 has a dominant role in ligand binding, but IFNAR-1 contributes to binding affinity and to differential ligand recognition. A panel of five monoclonal antibodies (mAb) to human IFNAR-1 (HuIFNAR-1) was produced and characterized. The reactivity of each mAb toward HuIFNAR-1 on native and transfected cells and in Western blot and ELISA formats was determined. In functional assays, one mAb, EA12, blocked IFN-a2 binding to human cells and interfered with Stat activation and antiviral activity. Epitopes for the mAb were localized to subdomains of the HuIFNAR-1 extracellular domain by differential reactivity of the mAb to a series of human/bovine IFNAR-1 chimeras. The antibody EA12 seems to require native HuIFNAR-1 for reactivity and does not map to a single subdomain, perhaps recognizing an epitope containing noncontiguous sequences in at least two subdomains. In contrast, the epitopes of the non-neutralizing mAb FB2, AA3, and GB8 mapped, respectively, to the first, second, and third subdomains of HuIFNAR-1. The mAb DB2 primarily maps to the fourth subdomain, although its reactivity may be affected by other determinants.     

Characterization of novel neutralizing monoclonal antibodies specific to human neurturin

Neurturin (NTN) a structural and functional relative of glial cell line-derived neurotrophic factor, was originally identified based on its ability to support the survival of sympathetic neurons in culture. Similar to glial cell line-derived neurotrophic factor (GDNF), Neurturin has been shown to bind to a high affinity glycosylphosphatidylinositol (GPI)-linked receptor (GFRalpha2) and induce phosphorylation of the tyrosine kinase receptor Ret, resulting in the activation of the mitogen activated protein kinase (MAPK) signalling pathway. A panel of six novel murine monoclonal antibodies (MAbs) specific to human Neurturin has been developed and characterized. Four of the MAbs tested inhibit, to varying degrees, binding of NTN to the GPI-linked GFRalpha2 receptor. Three MAbs cross-react with the murine homolog. These antibodies have been shown to be useful reagents for Western blotting, immunohistochemistry, and also for the development of a sensitive, quantitative enzyme-linked immunosorbent assay (ELISA) for human NTN. Novel, specific MAbs with varying epitope specificities and blocking activity will be valuable tools for both the in vitro and in vivo characterization of NTN and its relationship to the GFRalpha2 and Ret receptors.     

Characterization and potential diagnostic application of monoclonal antibodies specific to rabies virus

Objective

Rabies is invariably a fatal encephalomyelitis that is considered to be a serious public health problem. It is necessary to develop standard rabies virus diagnostic tools, especially for diagnosing the strains prevalent in China.

Methods

Monoclonal antibodies (MAbs) specific to rabies virus were produced and characterized by enzyme linked immunosorbent assay (ELISA), isotyping, affinity assay, immunofluorescence assay (IFA), and immunocytochemistry. The MAb, whose affinity was higher for antigen, was used to establish an antigen capture-ELISA (AC-ELISA) detection system and test the efficiency by using clinical samples.

Results

The heavy chain subclasses of two MAbs were all determined to be IgG2a. The 3C7 MAb showed stronger reactivity with rabies virus protein than the 2C5 MAb in an ELISA analysis, whereas the 3C7 MAb showed the highest affinity for antigen. IFA and immunocytochemistry results also indicated that the two MAbs could recognize rabies virus protein in its native form in cell samples. Data obtained using clinical samples showed that rabies virus could be detected by AC-ELISA detection system using the 3C7 MAb.

Conclusion

It was potentially useful for the further development of highly sensitive, easily handled, and relatively rapid detection kits/tools for rabies surveillance in those areas where rabies is endemic, especially in China.     

Characterization of monoclonal antibodies specific for the Merkel cell polyomavirus capsid

Merkel cell polyomavirus (MCV) has been implicated as a causative agent in Merkel cell carcinoma. Robust polyclonal antibody responses against MCV have been documented in human subjects, but monoclonal antibodies (mAbs) specific for the VP1 capsid protein have not yet been characterized. We generated 12 mAbs capable of binding recombinant MCV virus-like particles. The use of a short immunogenic priming schedule was important for production of the mAbs. Ten of the 12 mAbs were highly effective for immunofluorescent staining of cells expressing capsid proteins. An overlapping set of 10 mAbs were able to neutralize the infectivity of MCV-based reporter vectors, with 50% effective doses in the low picomolar range. Three mAbs interfered with the binding of MCV virus-like particles to cells. This panel of anti-capsid antibodies should provide a useful set of tools for the study of MCV.     

Characterization of human p53 antigens employing primate specific monoclonal antibodies

p53 is a cellular protein whose levels are some 1500-2000 times higher in adenovirus and SV40-transformed human cell lines than in homologous nontransformed cells. Monoclonal antibodies have been produced that detect p53 of primate origin but not of rodent origin. These monoclonal antibodies have been employed to study the properties of p53 antigens from human cell lines. Human p53 proteins of at least five different apparent molecular-weight classes in SDS-polyacrylamide gels have been detected. In some cell lines, at least two distinct molecular-weight species are expressed and these two forms have similar or identical partial peptide maps. Both molecular-weight forms can be resolved into seven or eight species upon isoelectric focusing in a two-dimensional gel system. There is also some indication of differences in the partial peptide maps of human p53 antigens derived from different human transformed cell lines. A radioimmunometric assay was employed to study the steady-state levels of oligomeric p53 in normal and transformed cell lines. Antibody affinity chromatography has been employed to purify p53 protein which was then used to quantitate the steady-state levels of p53 in different human cell lines. Normal cells had little or no detectable p53 antigen. Transformed cells or tumor-derived cell lines varied between no detectable p53 protein and 450 micrograms of p53 protein/g of cellular protein (in SV80 cells). There was a great diversity in the levels of p53 antigen in human cells. SV40- and adenovirus-transformed cells had by far the highest levels of p53 antigen. These are the viruses whose tumor antigens have been shown to be associated in an oligomeric complex with p53 in transformed cells. Eleven out of fifteen human tumor derived or transformed cell lines contained greater than five-fold higher levels of p53 antigen than normal human cells.     

Epitope mapping of anti-breast and anti-ovarian mucin monoclonal antibodies.

Anti-breast cancer antibodies (BC2, HMPV and 4B6) and an anti-ovarian cancer antibody (OM1) were found to react with mucins--indeed with the protein core encoded by the MUC1 gene. This gene contains a VNTR (variable number of tandem repeats) encoding a 60 bp (= 20 amino acids) repeat sequence and within this amino acid sequence SAPDTRPAP was predicted, by hydrophilicity analysis, to be the immunogenic peptide sequence. The four antibodies were shown to react with MUC1 VNTR encoded peptides in direct binding and inhibition studies. The precise reactivity of the 4 mAbs was mapped using ELISA in both solid and liquid phase, and demonstrated the epitopes to be: APDTR (BC2 and HMPV), PDTR (4B6) and DTRPA (OM1). By using the pepscan method, the epitopes were shorter (PDTR, DTR and DTRP). However when these short peptides (except DTR) were synthesized they did not react; flanking amino acids are needed for the epitopes. Clearly several different methods should be used to define the reactive epitope. Within (S)APDTR, major amino acid substitutions could be made--even of three to four amino acids without altering antibody binding, provided that P and R were not substituted. It was of interest that an anti-ovarian cancer antibody gave similar anti-peptide reactions to the anti-breast cancer antibodies; apparently MUC1 peptides in ovarian cancer are the same as in breast cancer.     

Characterization and application of monoclonal antibodies specific to West Nile virus envelope protein

Recent epidemics of West Nile virus (WNV) around the world have been associated with significant rates of mortality and morbidity in humans. To develop standard WNV diagnostic tools that can differentiate WNV from Japanese encephalitis virus (JEV), four monoclonal antibodies (MAbs) specific to WNV envelope (E) protein were produced and characterized by isotyping, reactivity with denatured and native antigens, affinity assay, immunofluorescence assay (IFA), and epitope competition, as well as cross-reactivity with JEV. Two of the MAbs (6A11 and 4B3) showed stronger reactivity with E protein than the others (2F5 and 6H7) in Western blot analysis. 4B3 could bind with denatured antigen, as well as native antigens in indirect ELISA, flow cytometry analysis, and IFA; whereas 2F5 showed highest affinity with native antigen. 4B3 and 2F5 were therefore used to establish an antigen capture-ELISA (AC-ELISA) detection system. The sensitivity of this AC-ELISA was 3.95 TCID(50)/0.1 ml for WNV-infected cell culture supernatant. Notably, these MAbs showed no cross-reactivity with JEV, which suggests that they are useful for further development of highly sensitive, easy handling, and less time-consuming detection kits/tools in WNV surveillance in areas where JEV is epidemic.     

Characterization of monoclonal antibodies against prostate specific antigen produced by genetic immunization

Prostate specific antigen (PSA) is the most important marker for prostate cancer. Antibodies against minor variants of PSA may be useful in the development of novel diagnostic tests for prostate cancer, but it has been difficult to produce such antibodies by protein immunization. In this study, we have compared the characteristics of monoclonal antibodies (MAbs) obtained by genetic immunization with those obtained by protein immunization. The whole coding region of PSA-cDNA was cloned in a mammalian expression vector pCDNA-3. Six mice were immunized four times by intra-muscular (i.m.) injection of the PSA-pCDNA3 plasmid. The MAbs produced were characterized with respect to subclass, epitope specificity, binding to various molecular forms of PSA and affinity. After intra-muscular injection of DNA, anti-PSA antibodies were detected in the serum of all mice, but the antibody titers were markedly lower than after protein immunization. After fusion of the spleen cells from the mice, five hybridomas producing MAbs to PSA were obtained. The MAbs were of IgG1 and IgG2a isotype and they all recognized equally different forms of free PSA, namely enzymatically active, nicked and proPSA. Epitope mapping showed that these MAbs reacted with the same antigenic regions as those obtained by protein immunization. Thus, genetic immunization leads to production of anti PSA MAbs with similar characteristics to those obtained by immunizing with PSA protein. As applied in the present study, it is less efficient than protein immunization, but it is a useful technique when the antigen is not available in the quantities needed for immunization.     

Characteristics of gut specific monoclonal antibodies

Eight distinct murine monoclonal antibodies that are directed to normal human mouse small intestinal epithelial cells have been characterized. Three of these called TPNG 2, 20 and 43 were directed to goblet cells and mucin components. One antibody, TPNC 10, was directed to basilar crypt cell inclusions and was specific for the small bowel and possible progenitor cells. Another antibody, TPNC 30, was directed to the microvilli of the brush border. Finally, three antibodies, TPNC 50, 51 and 52 reacted with epithelial cell membranes. These three antibodies reacted with intestinal epithelial cell membranes but also cross-reacted with hepatocyte cytoplasm. The availability of intestinal specific monoclonal reagents will be of significant interest in studies of normal and abnormal intestinal physiology.     

Characterization of monoclonal antibodies specific for feline serum amyloid (SAA) protein

Serum amyloid A (SAA) has been characterized as an inflammatory marker in many species. In this study, we have developed and characterized monoclonal antibodies (MAbs) against feline SAA (fSAA) derived from culture hybridomas. These hybridomas were produced from the fusion of Balb/c-derived myeloma s/p20-Ag14 and splenocytes from mice immunized with purified recombinant feline SAA (rfSAA). Six hybridomas secreting MAbs, M2, M5, M7, M8, M13, and M15, were selected and subcloned on the basis of their specificity to rfSAA by enzyme-linked immunoabsorbent assay (ELISA), and confirmed based on their specificity to rf-SAA by immunoblot analysis. Out of six clones, two clones (M5 and M7) showed higher reactivity with rf-SAA, and were selected for further analysis of ELISA additivity and Western blot cross-reactivity tests. As a result, M5 and M7 clones recognized the same or excessively near epitopes on rfSAA and reacted with rfSAA, fSAA and equine recombinant SAA, but showed no reaction with human recombinant SAA. Because of their specificity, these MAbs may be usefully applied in studying the measurement of SAA concentration in cat serum.     

Characterization of monoclonal antibodies specific for equine homologues of CD3 and CD5.

Two monoclonal antibodies (mAb), UC F6G-3 and UC F13C-5, were characterized as being specific for the apparent equine homologues of CD3 and CD5, respectively. Both antibodies exhibited characteristics of pan-T-lymphocyte markers based upon immunohistology and two-colour flow cytometry. UC F6G-3 precipitated a complex of proteins (up to seven) with molecular weights ranging from 18,000 to 42,000, similar to the human and murine CD3 complex. Upon further dissociation of the precipitated complex, two proteins were identified with molecular weights of 22,000 and 27,000. Immobilized UC F6G-3 was effective at inducing interleukin-2 receptor (IL-2R) expression on T lymphocytes, a feature consistent with antibodies specific for the epsilon chain of human and murine CD3. Three populations of cells in the thymus were distinguishable by UC F6G-3 target antigen density, suggesting increasing stages of T-cell maturation. UC F13C-5 precipitated a 67,000 MW protein, consistent with reported values for CD5 in multiple species. While this antibody exhibited characteristics of a pan-T-cell marker, low numbers of B lymphocytes also expressed the target antigen. Phorbol esters induced variable increases in target antigen density on B lymphocytes. These two antibodies, taken together with the few equine CD markers currently available, represent a substantial resource for further defining the equine immune system in health and disease.     

Characterization of Hantaviruses using monoclonal antibodies

The antigenic relations among nine Hantaviruses from seven different geographic areas were examined in indirect fluorescent antibody test with 12 monoclonal antibodies. Ten monoclonals were prepared against Hantaan virus strain 76-118 and two against Hantavirus B-1, a virus isolated from a tumour in a laboratory rat. Four different serotypes have been differentiated: H?lln?s, Hantaan, Prospect Hill and Rat strain like. Two monoclonal antibodies were able to react with all nine Hantaviruses studied. Rat strain- and Hantaanvirus-specific monoclonal antibodies were documented.     

Purification of anti-epithelial mucin monoclonal antibodies by epitope affinity chromatography.

Monoclonal antibodies against the protein core of epithelial mucins have been found to react with the immunodominant sequence P D T R P A P (Burchell et al., 1989; Price et al., 1990a). Two immunoadsorbent matrices were prepared by linking the peptide A P D T R P A P G to CNBr-activated Sepharose and by linking the peptide C A P D T R P A P G to activated thiol-Sepharose, so that each immunoadsorbent contained the immunodominant motif. Anti-epithelial mucin antibodies (anti-breast carcinoma antibodies, anti-purified mucin antibodies and anti-human milk fat globule antibodies) were examined for reactivity with these preparations. The initial tests indicated that the substituted CNBr-activated Sepharose displayed lower non-specific antibody binding and this matrix was selected for further investigation. The anti-mucin antibodies were shown to react specifically with this affinity matrix and irrelevant antibodies failed to bind. A Sepharose-peptide immunoadsorbent column was examined for its capacity to purify several of these anti-mucin antibodies and it was determined that this procedure was highly efficient--purified IgG and IgM antibodies could be isolated from either hybridoma tissue culture supernatants or ascitic fluids. The capacity of the column was in excess of 40 mg antibody protein per ml of gel for the IgG3 antibody, C595 (anti-urinary mucin) and at least 10 mg antibody protein per ml of gel for the IgM antibody, NCRC-11 (anti-breast carcinoma). The procedure described permits the efficient purification of anti-mucin antibodies and provides a product which would be suitable for further investigations requiring highly immunoreactive antibodies (e.g., for radioimmunotherapy or immunoscintigraphy in patients with malignant disease).     

Intracellular variation of rat intestinal mucin granules localized by monoclonal antibodies

Monoclonal antibodies produced against rat small intestinal mucins were utilized to study variability of stored mucin granules within rat ileal goblet cells. Eleven antibody-secreting hybridoma cultures were produced; six of these uniformly labeled stored mucin granules in virtually all goblet cells, suggesting that some antigenic features are common to all granules. The other five stained goblet cells in the rat small intestinal epithelium nonuniformly. R803, R805, and R807 localized within almost all goblet cells but revealed differential labeling of centrally and peripherally located mucin granules. R804 uniformly labeled the mucin granules of most villous goblet cells; some of the crypt goblet cells were uniformly labeled, but the majority were only partially labeled, resulting in a mottled staining pattern. R808 stained only a small portion of crypt goblet cells; there is, however, an increase in both number of goblet cells labeled and in uniformity of staining of the stored granule mass from the base of the crypt to the surface, resulting in uniform labeling of virtually all goblet cells at the villus tip. This study demonstrates for the first time that rat small intestinal mucin granules are immunologically heterogeneous and nonuniformly distributed within the epithelium. Additionally, staining patterns within the stored granule mass suggest that structurally distinct subpopulations of mucin granules may exist within a single goblet cell.     

Characterization of three human monoclonal antibodies specific for polymorphic class I and class II HLA antigens

Three human monoclonal antibodies were derived from a single polytransfused patient awaiting renal transplantation. In microcitotoxicity assays, the patient's serum displayed strong positive reactions against> 90% of a panel of cells representing the known HLA specificities. The donor's peripheral blood lymphocytes were infected with Epstein-Barr virus, cloned, and supernatants of the virus transformed cultures were screened for the presence of IgG antilymphocyte reactivity utilizing an enzyme-linked immunosorbent assay method. Positive cultures were recloned and fused with the human-mouse heteromyeloma SHM. Supernatants from three clones were selected for alloreactivity and characterized by indirect immunofluorescent staining and fluoractivated cell sorter analysis on homozygous typing cells, including those from the Tenth International Histocompatibility Workshop core panel and on cell lines derived from selected families. Data obtained demonstrate that two human monoclonal antibodies have DQw1 specificity, one of them being reactive against several DQw7-positive cell lines, while one monoclonal antibody is specific for the A2 + A28 class I MHC antigens. Anti-DQw1 antibodies were of different light-chain subtypes.     

Production and characterization of highly specific anti-methotrexate monoclonal antibodies

Spleen cells of a Biozzi HR mouse immunized with a bovine serum albumin-methotrexate conjugate were fused with P3-X63-Ag8.653 mouse myeloma cells. Twenty-three monoclonal antibodies (MAbs), selected by indirect ELISA, were produced and partially characterized. Using methotrexate (MTX) and eight structurally related compounds, binding specificities of the MAbs were assessed by inhibition enzyme immunoassay. All the MAbs had very low affinity for folic acid and its analogs and for the major MTX metabolite 7-hydroxymethotrexate. Using a computer cluster analysis program based on the binding specificities, the MAbs were divided into three groups. The thirteen MAbs in group I recognized primarily the pteridine portion of the MTX molecule; the eight group II MAbs recognized the benzene ring as well as the pteridine structure. The two MAbs in group III poorly distinguished between the different parts of the MTX molecule. The apparent equilibrium association constants of the anti-MTX MAbs in groups I, II, and III ranged from 7 x 10(9) to 3 x 10(8) M-1 (except for 1 MAb), from 5 x 10(7) to 6 x 10(6) M-1 (except for 2 MAbs), and from 1 x 10(6) to 3.5 x 10(5) M-1, respectively.     

Characterization of monoclonal antibodies against human lactoferrin

The iron-binding glycoprotein human lactoferrin (hLF) is involved in the host defense against infection and is a modulator of inflammatory reactions. We generated monoclonal antibodies (mAbs) to hLF as tools to assist both structure-function studies and the development of recombinant human lactoferrin for applications in human health care. Binding experiments with ten distinct anti-hLF mAbs to tryptic and recombinant hLF fragments in ELISA and/or on immunoblots revealed that five mAbs bound to conformational epitopes residing in the N-lobe (residues 1 to 334), whereas the other five bound to C-lobe conformational epitopes (residues 335 to 692). None of the mAbs bound to hLF denatured upon reduction. Monoclonal antibody E11 appeared to bind to the arginine-rich N-terminus of hLF, which is the binding site for heparin, bacterial lipopolysaccharide, human lysozyme, DNA and receptors. The dissociation constant of the distinct mAbs for hLF ranged from 0.5 to 18 nM, without differences in affinity for unsaturated or iron-saturated hLF, indicating that the conformational changes subject to incorporation of iron do not seem to affect the exposure and/or conformation of the antibody epitopes. The mAbs did not bind to human transferrin, a protein closely related to hLF in size, primary amino acid sequence and structure. Two C-lobe specific mAbs, E2 and E8, cross-reacted with bovine and/or porcine lactoferrin, indicating that human, bovine and porcine lactoferrin share antigenic determinants. This panel of mAbs will be used to develop quantitative and qualitative immunoassays for hLF and to delineate which regions of hLF are relevant to its anti-infective and anti-inflammatory properties.     

Characterization of Zaire ebolavirus glycoprotein-specific monoclonal antibodies

Zaire ebolavirus (ZEBOV) can be transmitted by human-to-human contact and causes acute haemorrhagic fever with case fatality rates up to 90%. There are no effective therapeutic or prophylactic treatments available. The sole transmembrane glycoprotein (GP) is the key target for developing neutralizing antibodies. In this study, recombinant VSVΔG/ZEBOVGP was used to generate monoclonal antibodies (MAbs) against the ZEBOV GP. A total of 8 MAbs were produced using traditional hybridoma cell fusion technology, and then characterized by ELISA using ZEBOV VLPs, Western blotting, an immunofluorescence assay, and immunoprecipitation. All 8 MAbs worked in IFA and IP, suggesting that they are all conformational MAbs, however six of them recognized linearized epitopes by WB. ELISA results demonstrated that one MAb bound to a secreted GP (sGP 1-295aa); three bind to a part of the mucin domain (333-458aa); three MAbs recognized epitopes on the C-terminal domain of GP1 (296-501aa); and one bound to full length GP (VLPs/GP1,2 ΔTm). Using a mouse model these MAbs were evaluated for their therapeutic capacity during a lethal infection. All 8 MAb improved survival rates by 33%-100% against a high dose lethal challenge with mouse-adapted ZEBOV. This work has important implications for further development of vaccines and immunotherapies for ZEBOV infection.