Human antibodies and hybridomas最新文献

{"title":"Lung cancer-reacting human recombinant antibody AE6F4: potential usefulness in the sputum cytodiagnosis.","authors":"M Shoji,&nbsp;S Kawamoto,&nbsp;K Seki,&nbsp;K Teruya,&nbsp;Y Setoguchi,&nbsp;K Mochizuki,&nbsp;M Kato,&nbsp;S Hashizume,&nbsp;T Hanagiri,&nbsp;T Yoshimatsu,&nbsp;K Nakanishi,&nbsp;K Yasumoto,&nbsp;A Nagashima,&nbsp;H Nakahashi,&nbsp;T Suzuki,&nbsp;T Imai,&nbsp;S Shirahata,&nbsp;K Nomoto,&nbsp;H Murakami","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>Human monoclonal antibody (hMAb) AE6F4 has been shown to be potentially useful for immunocytological detection of lung cancer cells in sputum. By recombinant DNA technology, IgM type hMAb AE6F4 was switched to lgG. The IgG mimic recombinant AE6F4 antibody expression plasmid was assembled using the antibody heavy chain gene, which ligated the gene encoding VH and CH1(mu) domains of hMAb AE6F4 heavy chain to the gene encoding CH2(gamma 1) and CH3(gamma 1) domains of human IgG heavy chain, and the antibody light chain gene of hMAb AE6F4. The recombinant antibody expressed by baby hamster kidney (BHK)-21 cells showed molecular size equivalence to IgG, and consisted of human mu-gamma hybrid heavy and kappa light chains. The immunological specificity of the recombinant antibody was the same as that of hMAb AE6F4 by immunoblotting analysis to the 14-3-3 protein, the putative antigen of hMAb AE6F4, and by immunohistochemical and immunocytological analyses using tissue sections and sputa of lung cancer patients. The transfected BHK-21 cells produced the recombinant antibody persistently and the productivity was greater than 20 times that by human-human hybridoma producing hMAb AE6F4.</p>","PeriodicalId":77166,"journal":{"name":"Human antibodies and hybridomas","volume":"7 1","pages":"27-36"},"PeriodicalIF":0.0,"publicationDate":"1996-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19851347","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Membranous nephropathy in primary antiphospholipid syndrome: description of a case and induction of renal injury in SCID mice.","authors":"Y Levy,&nbsp;L Ziporen,&nbsp;B Gilburd,&nbsp;J George,&nbsp;S Polak-Charcon,&nbsp;H Amital,&nbsp;J Cledes,&nbsp;P Youinou,&nbsp;Y Shoenfeld","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>Primary antiphospholipid syndrome (PAPS) is a recently recognized clinical entity encompassing the combination of thromboembolic phenomena, thrombocytopenia and recurrent abortions in the presence of antiphospholipid antibodies. We present a patient with PAPS accompanied by renal involvement, manifested as membranous nephropathy, as proven by a renal biopsy. To investigate further the possible association between PAPS and the renal lesions we attempted to induce similar renal manifestations by transferring peripheral blood lymphocytes (PBL) from this patient to severe combined immunodeficiency (SCID) mice. The mice transfused with PBL from the affected patient exemplified antiphospholipid antibodies (aPL) following which a renal lesion consistent with the human membranous nephropathy lesion was precipitated. This study substantiates the role of aPL as possible inducers of renal damage.</p>","PeriodicalId":77166,"journal":{"name":"Human antibodies and hybridomas","volume":"7 3","pages":"91-6"},"PeriodicalIF":0.0,"publicationDate":"1996-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"20013637","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A rapid method for purification of monoclonal human IgM from mass culture.","authors":"H P Vollmers,&nbsp;E Wozniak,&nbsp;E Stepien-Bötsch,&nbsp;U Zimmermann,&nbsp;H K Müller-Hermelink","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>We describe in this paper the rapid and cheap purification of human immunoglobulin M from hybridoma supernatant of mass culture. The method consists of two steps: 1. concentration of supernatant by ultrafiltration and 2. dialysis against distilled water, pH 6.4. To produce the supernatant, the hybridomas are grown in RPMI media with fetal calf serum (10% FCS) in 250 ml flasks under normal tissue culture conditions. More than 14 mg IgM can be nearly selectively purified within 6 h from 5 l of antibody containing hybridoma supernatant. Most of the IgM molecules stay in a penta- and monomeric form and are positive in physiological and immunohistochemical studies.</p>","PeriodicalId":77166,"journal":{"name":"Human antibodies and hybridomas","volume":"7 1","pages":"37-41"},"PeriodicalIF":0.0,"publicationDate":"1996-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19851348","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Cross reactive anti-tetanus and anti-melittin Fab fragments by phage display after tetanus toxoid immunisation.","authors":"M. Vogel, L. Lai, M. Rudolf, V. Curcio‐Vonlanthen, S. Miescher, B. Stadler","doi":"10.3233/HAB-1996-7102","DOIUrl":"https://doi.org/10.3233/HAB-1996-7102","url":null,"abstract":"Amino acid sequence homology between tetanus toxoid, a common vaccine and melittin, a bee venom allergen provided us with two antigen models for studying the production of cross reactive antibodies after immunisation. Analysis of the serum of an atopic patient recently boosted with tetanus toxoid revealed the presence of anti-melittin and anti-tetanus antibodies. From this donor a phage display Fab library was constructed five days after vaccination. Screening of this library either with melittin alone or with tetanus toxoid followed by melittin allowed the isolation of Fab fragments specific to melittin but also Fab fragments which cross react with melittin and tetanus toxoid. Amino acid analysis revealed diversity in the heavy and the light Ig chains of the melittin specific and of the cross reactive clones. Interestingly we found that the light chain recognised melittin whereas the heavy chain preferentially bound to tetanus toxoid suggesting that cross reactivity may be due to the different binding specificities of the individual Ig chains.","PeriodicalId":77166,"journal":{"name":"Human antibodies and hybridomas","volume":"7 1 1","pages":"11-20"},"PeriodicalIF":0.0,"publicationDate":"1996-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3233/HAB-1996-7102","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"69906018","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Humanization of an anti-human IL-6 mouse monoclonal antibody glycosylated in its heavy chain variable region.","authors":"K Sato,&nbsp;T Ohtomo,&nbsp;Y Hirata,&nbsp;H Saito,&nbsp;T Matsuura,&nbsp;T Akimoto,&nbsp;K Akamatsu,&nbsp;Y Koishihara,&nbsp;Y Ohsugi,&nbsp;M Tsuchiya","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>Interleukin-6 (IL-6) inhibitors are good potential therapeutic agents in human patients, and anti-IL-6 antibodies are among the best candidates. Here, we have successfully humanized mouse monoclonal antibody SK2, which specifically binds to IL-6 and strongly inhibits IL-6 functions. Since this antibody possesses N-linked carbohydrates on Asn-30 of VH region, which seems to be very close to an antigen-binding site, influence of these carbohydrates on antigen-binding was investigated. A biosensor study showed that the mouse SK2 Fab and its deglycosylated fragments had almost equal Kd (Kon/Koff), 26.8 nM (1.05 x 10(6)/2.81 x 10(-2)) and 24.7 nM (1.28 x 10(6)/3.15 x 10(-2)), respectively. Furthermore, a mutant chimeric SK2 antibody, in which the N-glycosylation site was removed from the VH region, showed a Kd of 11 nM, almost similar to that of the original chimeric SK2 antibody, determined by Scatchard analysis with 125I-IL-6. These data indicate the carbohydrates of mouse SK2 VH region do not significantly influence antigen-binding activity. In the next step, two versions of each humanized SK2 VL and VH regions were carefully designed based on the amino acid sequences of human REI and DAW, respectively. Only one alteration, Tyr to Phe, was made at position 71 in the two light chains, according to the canonical residue for LI. A N-glycosylation site was introduced on the two heavy chains, by changing Ser to Asn at position 30. All four combinations of humanized light and heavy chains could bind to IL-6 as well as the chimeric SK2 antibody. The light chain first version, however, could not efficiently inhibit IL-6 binding to its receptor, indicating the importance of the LI loop conformation for the inhibitory activity of SK2 antibody. In contrast, both versions of the heavy chains were comparable, in yielding good humanized SK2 antibodies, suggesting that the glycosylation of the SK2 VH region has no influence in recreating a functional antigen-binding site in this humanization.</p>","PeriodicalId":77166,"journal":{"name":"Human antibodies and hybridomas","volume":"7 4","pages":"175-83"},"PeriodicalIF":0.0,"publicationDate":"1996-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"20088760","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Cross reactive anti-tetanus and anti-melittin Fab fragments by phage display after tetanus toxoid immunisation.","authors":"M Vogel,&nbsp;L Lai,&nbsp;M P Rudolf,&nbsp;V Curcio-Vonlanthen,&nbsp;S Miescher,&nbsp;B M Stadler","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>Amino acid sequence homology between tetanus toxoid, a common vaccine and melittin, a bee venom allergen provided us with two antigen models for studying the production of cross reactive antibodies after immunisation. Analysis of the serum of an atopic patient recently boosted with tetanus toxoid revealed the presence of anti-melittin and anti-tetanus antibodies. From this donor a phage display Fab library was constructed five days after vaccination. Screening of this library either with melittin alone or with tetanus toxoid followed by melittin allowed the isolation of Fab fragments specific to melittin but also Fab fragments which cross react with melittin and tetanus toxoid. Amino acid analysis revealed diversity in the heavy and the light Ig chains of the melittin specific and of the cross reactive clones. Interestingly we found that the light chain recognised melittin whereas the heavy chain preferentially bound to tetanus toxoid suggesting that cross reactivity may be due to the different binding specificities of the individual Ig chains.</p>","PeriodicalId":77166,"journal":{"name":"Human antibodies and hybridomas","volume":"7 1","pages":"11-20"},"PeriodicalIF":0.0,"publicationDate":"1996-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19851345","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Immunogenetics of human IgE.","authors":"R. Snow, C. Chapman, S. Holgate, F. Stevenson","doi":"10.3233/HAB-1996-7403","DOIUrl":"https://doi.org/10.3233/HAB-1996-7403","url":null,"abstract":"Immunoglobulin E plays a central role in mediating the pathology of allergic disease. Conversely, it is involved in the normal protective immune responses against parasite infection. Both these biological processes depend on interaction between the variable regions (VH and VL) of IgE antibodies and target antigen. It is now feasible to investigate the molecular nature of VH regions used to encode IgE at the genetic level. Using this technology to analyze IgE in patients with asthma has revealed features which may have relevance for allergic disease. First, preferential choice of VH genes, with dominance of the small VH5 family, particularly the VH32 gene, has been found. This may implicate a B cell superantigen (superallergen) selectively driving the use of these genes. Second, VH5 genes in IgE are somatically mutated with clear hot spots of mutational activity. Mutational hotspots, which are a feature of the VH5 gene, are supplemented in IgE by ongoing mutations which may be involved in affinity maturation. Third, a single B cell can switch to either IgE or IgG4, with both variants coexisting in blood. These findings may provide clues to the mechanism by which IgE is generated, and suggest options for therapeutic intervention.","PeriodicalId":77166,"journal":{"name":"Human antibodies and hybridomas","volume":"34 1","pages":"157-66"},"PeriodicalIF":0.0,"publicationDate":"1996-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3233/HAB-1996-7403","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"69906825","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Unsusceptibility of recombinant human Fc fragments of immunoglobulin E to thrombin.","authors":"T. Kamiya","doi":"10.3233/HAB-1996-7106","DOIUrl":"https://doi.org/10.3233/HAB-1996-7106","url":null,"abstract":"Human immunoglobulin E (IgE) contains a potential recognition sequence for thrombin protease cleavage at N-terminal end of C epsilon 3 domain responsible for binding to alpha chain of high affinity Fc receptor for IgE (Fc epsilon RI alpha), but it remains unknown for the enzyme susceptibility. The human Fc fragments of IgE (IgE-Fc), consisting of C epsilon 2' (Ala329)-C epsilon 3-C epsilon 4 chains (Fc') and of C epsilon 2' (Thr315-Ala329)-C epsilon 3-C epsilon 4 chains (F(c')2), were expressed in the mammalian COS and Chinese hamster ovary (CHO) cells by placing the IgE-Fc cDNA under the control of the cytomegalovirus (CMV) promoter. Under nonreducing condition F(c')2 was not cleaved by thrombin protease as well as native IgE. Neither treatment of F(c')2 with final 0.1% 2-mercaptoethanol at boiling point for 5 min, with a sulfhydryl-reactive biotin derivative, by which monomers in COS-derived F(c')2 preparations were biotinylated at Cys328, nor with neuraminidase, affected the accessibility to the enzyme. These results suggested that F(c')2, either dimeric or monomeric, had compact conformations.","PeriodicalId":77166,"journal":{"name":"Human antibodies and hybridomas","volume":"16 1","pages":"42-7"},"PeriodicalIF":0.0,"publicationDate":"1996-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3233/HAB-1996-7106","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"69906162","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Efficient in vitro affinity maturation of phage antibodies using BIAcore guided selections.","authors":"R. Schier, J. Marks","doi":"10.3233/HAB-1996-7302","DOIUrl":"https://doi.org/10.3233/HAB-1996-7302","url":null,"abstract":"Selection of higher affinity mutant phage antibodies has proven less than straightforward due to sequence dependent differences in phage antibody expression, toxicity to Escherichia coli, and difficulty in eluting the highest affinity phage. These differences lead to selection for increased levels of expression or decreased toxicity rather than for higher affinity. In this work, we demonstrate how surface plasmon resonance as employed in the BIAcore can be used to increase the efficiency of phage antibody selections, yielding greater increments in affinity from a single library. A mutant phage antibody library was created by randomizing nine amino acids located in the V(L) CDR3 of C6.5, a human scFv which binds the tumor antigen c-erbB-2 with a Kd of 1.6 x 10-8 M. The library was subjected to five rounds of selection in solution using decreasing concentrations of biotinylated c-erbB-2. After each round of selection, polyclonal phage were prepared and the rate of binding to c-erbB-2 determined in a BIAcore under mass transport limited conditions. Determination of the rate of binding permitted calculation of the concentration, and hence percent, of binding phage present. Results were used to select the antigen concentration for the next round of selection. To determine the optimal eluent, polyclonal phage was injected in a BIAcore and eluted using one of five different solutions (10 mM HCl, 50 mM HCl, 100 mM HCl, 100 mM triethylamine, 2.6 M MgCl2). Differences were observed in eluent efficacy, which was reflected in significant differences in the affinities of phage antibodies isolated from the library after a round of selection using the different eluents. Use of the BIAcore to determine the optimal eluent and guide the antigen concentration used for selection yielded a C6.5 mutant with a 16 fold reduction in Kd (Kd = 1.0 x 10-9 M). This represents at least a twofold greater increment in affinity than previously obtained from a single library of phage antibodies which bind antigens.","PeriodicalId":77166,"journal":{"name":"Human antibodies and hybridomas","volume":"7 3 1","pages":"97-105"},"PeriodicalIF":0.0,"publicationDate":"1996-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3233/HAB-1996-7302","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"69906228","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Short synthetic CDR-peptides forming the antibody combining site of the monoclonal antibody against RNA bacteriophage fr neutralize the phage activity.","authors":"J. Steinbergs, K. Kilchewska, U. Lazdina, A. Dishlers, V. Ose, M. Sällberg, A. Tsimanis","doi":"10.3233/HAB-1996-7303","DOIUrl":"https://doi.org/10.3233/HAB-1996-7303","url":null,"abstract":"The construction of a mouse hybridoma FR52 secreting neutralizing monoclonal antibody specific for RNA bacteriophages fr, MS2 and GA is reported. The genes encoding the variable domains of the monoclonal antibody FR52 heavy and light chains were cloned and sequenced and the corresponding complementarity determining region (CDR) peptides were chemically synthesized. The CDR-peptides were tested for their ability to neutralize the activity of RNA phage fr and related RNA phages MS2 and GA. The CDR-derived peptides H2, L2 and L3 interacted with the fr phage particles and neutralized fr phage activity. Two of these peptides--H2 and L3 also had the ability to neutralize partly the activity of related bacteriophage MS2, but L1 and especially L3 neutralize the activity of the RNA phage GA. These results provide an excellent system for further antibody-antigen interaction studies and raise the possibility that simple CDR-peptides may serve as a new class of anti-viral molecules.","PeriodicalId":77166,"journal":{"name":"Human antibodies and hybridomas","volume":"7 3 1","pages":"106-12"},"PeriodicalIF":0.0,"publicationDate":"1996-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3233/HAB-1996-7303","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"69906648","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}