Kinetic analysis of interactions between bispecific monoclonal antibodies and immobilized antigens using a resonant mirror biosensor

A resonant mirror biosensor (IAsys) protocol is described for the comparative kinetic analysis of the ability of monoclonal antibodies (Mabs) and bispecific antibodies (Babs) to bind immobilized antigens. The protocol has been optimized and validated using the panel of affinity-purified antibodies, including two parental Mabs, one specific to human immunoglobulin G (hIgG) and another specific to horseradish peroxidase (HRP), and a Bab derived thereof by cell fusion (anti-hIgG/HRP Bab). The real-time kinetic analysis of antigen–antibody interactions using this protocol allows to demonstrate the differences in the avidity of bivalently binding Mabs and monovalent Babs. As shown in our previous study [J. Immunol. Methods 261 (2002) 103], the observed equilibrium association constants (K_ass) determined by IAsys using this protocol yield figures almost overlapping with those obtained by solid-phase radioimmunoassay (RIA). The described protocol is suited for the investigation of the effects of valency on the binding properties of antibodies. It also may be applied for the selection of Mabs and Babs with desired features, for different fields of application.     

Characterization and application of a Listeria monocytogenes reactive monoclonal antibody C11E9 in a resonant mirror biosensor

Typical detection of Listeria monocytogenes involves selective enrichment, isolation and biochemical testing. Development of antibodies to Listeria species has improved detection; however, most antibodies detect all species of Listeria. A previously developed monoclonal antibody (MAb)-C11E9 was examined for its reaction to 13 L. innocua and 40 L. monocytogenes strains representing all 13 serotypes by ELISA. Absorbance values for L. monocytogenes strains were 0.44-3.58 and for L. innocua 0.22-1.44. ELISA reactions were divided into three arbitrary groups of high (Abs 1.0 or higher), intermediate (0.6-0.99) and low (0.18-0.59). Most L. monocytogenes strains (32/41, 78%) were in the high group while only 23% (3/13) of L. innocua were in the same group. In the Western blot assay, antibody reacted with phosphate-buffered saline (PBS) extracted protein preparations of 52, 66 and 97 kDa. Ribopattern of all strains was analyzed and no clear relationship was observed for antibody reaction and ribotype of a given strain. MAb C11E9 was used in a resonant mirror biosensor (IAsys sensor), but failed to detect any viable intact L. monocytogenes cells at levels as high as 10(8) cells/ml; however, it showed binding (85-150 arc/s) with the surface protein preparations containing the 97-, 66- and 52-kDa proteins at 208 mug/ml. Binding kinetics of L. monocytogenes and L. innocua surface protein extracts showed significantly (p<0.05) higher responses than the three other Listeria species (L. ivanovii, L. welshimeri and L. grayi), which could be detected in 10-20 min. These data corroborate with ELISA results. In summary, this study suggest that MAb-C11E9 is suitable for detection of all serotypes of L. monocytogenes despite cross-reaction with L. innocua and could be used for detection of soluble protein extracts in the resonant mirror (IAsys) biosensor.     

Epitope analysis of the Goodpasture antigen using a resonant mirror biosensor

We have used a new technique for studying molecular interactions—a resonant mirror biosensor—to identify B cell epitopes within the Goodpasture antigen, which has recently been identified as the non-collagenous domain of the α3-chain of type IV collagen (α3(IV)NC1). Recombinant antigen (r-α3) was immobilized onto the sensing surface of a sample cuvette, and the binding of patients’ autoantibodies or a MoAb to the Goodpasture antigen was followed in real time. All patients’ sera bound r-α3 in this system, while control sera did not bind. A MoAb inhibited the binding of all patients’ autoantibodies to r-α3, from 27% to 90% (mean inhibition 60%), and patients’ sera cross-inhibited the binding of each other to the antigen. Binding was inhibited by pre-incubation of autoantibody with both native sheep α3(IV)NC1 and purified human α3(IV)NC1 monomers. Inhibition experiments using soluble overlapping peptides from human α3(IV)NC1 identified putative B cell epitopes. These results suggest that there is a major immunodominant epitope on the Goodpasture antigen, and that there is very limited heterogeneity in the autoantibody response in Goodpasture’s disease. The resonant mirror biosensor can be successfully used to monitor antibody–antigen binding using polyclonal sera, and to map epitopes on autoantigens.     

Analysis of the binding of bispecific monoclonal antibodies with immobilized antigens (human IgG and horseradish peroxidase) using a resonant mirror biosensor

The interaction between two monoclonal antibodies (mAbs) and their corresponding bispecific antibody (bAb) with immobilized antigens has been examined using a resonant mirror biosensor (IAsys). BAbs were produced by cell fusion. The analysed panel of affinity-purified antibodies included two parental mAbs, one specific to human IgG (hIgG), and another specific to horseradish peroxidase (HRP), and a bAb derived thereof (anti-hIgG/HRP). The real-time analysis showed the drastic differences in the avidity of bivalent anti-HRP mAbs and anti-HRP shoulder of bAbs. Thus, the observed equilibrium association constant (K(ass)) of anti-HRP mAbs was about 50 times higher that of anti-HRP shoulder of bAbs. The ratio of association rate constants (k(ass)) of mAbs and bAbs was about two, due to the statistical factor of two binding sites per bivalent antibody molecule. However, the dissociation rate constant (k(diss)) of anti-HRP shoulder of bAbs was 21 times higher k(diss) of anti-HRP mAbs. The comparison with the theoretical model shows that these observations are consistent only with a situation in which bivalent binding of mAbs with immobilized HRP predominates over monovalent binding. On the contrary, the second parental mAb (anti-hIgG) did not show the increase in avidity due to bivalent binding, compared to the anti-hIgG shoulder of bAbs, suggesting that this mAb was bound monovalently to immobilized hIgG. The K(ass) values determined by solid-phase radioimmunoassay (RIA) yielded figures almost overlapping with those obtained by IAsys. The results of the comparison of bAbs and mAbs are discussed from the viewpoint of the use of bAbs in heterogeneous systems. On the other hand, these data demonstrate that real-time analysis of antibody binding parameters in IAsys biosensor is valuable for the selection of mAbs and bAbs with desired features, for different fields of application.     

Rapid immunotyping of Chlamydia trachomatis with monoclonal antibodies in a solid-phase enzyme immunoassay.

The technical complexity of determining the serovar of Chlamydia trachomatis strains has limited the use of serotyping in clinical and epidemiologic studies. We developed a simple method for rapidly serotyping isolates of C. trachomatis by using monoclonal antibodies in a dot-enzyme-linked immunosorbent assay (ELISA) system. Isolates were passaged three to six times in shell vial cultures to greater than 50% monolayer infection, and chlamydial elementary bodies were isolated by sonication and microcentrifugation. Chlamydial antigen was spotted onto a series of replicate nitrocellulose membrane patches and reacted with C. trachomatis-specific monoclonal antibodies. Bound antibody was detected visually by a color reaction by using peroxidase-conjugated anti-mouse immunoglobulins. This method can be routinely applied to 60 or more specimens concurrently. We compared dot-ELISA serotyping with monoclonal antibody microimmunofluorescence serotyping of 124 clinical C. trachomatis isolates and found that dot-ELISA has sensitivity and serotyping accuracy comparable to that of monoclonal antibody microimmunofluorescence.     

生物传感器对抗戊型肝炎病毒单克隆抗体部分特性的研究

目的　应用生物传感器对新制备的抗戊型肝炎病毒单克隆抗体 (mAb)的亲和力、Ig亚类 (型 )及抗原结合的动力学进行研究。方法　用HEVORF2区基因工程重组蛋白NE2 ,免疫BALB/c小鼠 ,经杂交瘤技术制备mAb ,采用ELISA、Westernblot和生物传感器鉴定其有关特性。结果获得 12株可稳定分泌抗NE2mAb的杂交瘤细胞系。用ELISA及生物传感器等鉴定各株mAb ,分别为IgM和IgG1、IgG2a,轻链均为κ型。其中在用ELISA法对mAb3F5亚类鉴定过程中 ,发现其与HRP GAMIgG2a和HRP GAMIgM均有反应 ,生物传感器鉴定其为IgM。Westernblot验证各株mAb的特异性 ,同时各株mAb对于不同聚合形式的NE2蛋白的反应性有一定的差别。应用生物传感器测定了 4株mAb的KD 值 ,mAb 8C11为 4 .36× 10 7,mAb8H3为 1.5 3× 10 5,mAb 13D8为 1.2 1× 10 6,mAb 16D7为8.0 3× 10 7。从生物传感器测得的各株mAb与NE2的结合、解离过程中发现 ,mAb 8H3与NE2可快速结合、快速解离 ,其它 3株mAb结合稳定。结论　经多种方法鉴定 ,所获得的 12株杂交瘤细胞分泌的抗体 ,为抗HEVORF2区段的特异性抗体     

Diversity of anti-HLA-DR antibodies elicited by distinct anti-idiotypic monoclonal antibodies recognizing idiotopes co-expressed by the immunizing monoclonal antibody.

Analysis at the clonal level of the idiotypic network has identified differences in fine specificity between antigen-binding anti-anti-idiotypic (anti-anti-id) monoclonal antibody (mAb) and the original mAb as well as among antigen-binding anti-anti-idiotypic (anti-id) mAb. However, the diversity of humoral immune responses elicited by anti-id mAb recognizing idiotopes co-expressed on the immunizing mAb has not been analysed. Since this information may contribute to our understanding of the role of anti-id antibodies in the generation of diversity in the course of an immune response, we have compared the fine specificity and idiotype profile of two subsets of anti-HLA-DR mAb generated with the anti-id mAb F5-444 and F5-830. The latter mAb recognize idiotopes co-expressed in the antigen-combining site of the immunizing anti-HLA-DR1,4,w14,w8,9 mAb AC1.59. These investigations showed that: (1) the two subsets of anti-HLA-DR mAb overlap only partially in their reactivity patterns with HLA-DR+ cells; (2) both subsets of anti-HLA-DR mAb recognize spatially close epitopes; (3) each subset of anti-HLA-DR mAb has unique reactivity patterns with soluble HLA-DRw16 and DRw17 antigens; and (4) each subset of anti-anti-id mAb displays a distinct idiotype profile. The subtle differences in the fine specificity and idiotype profile of the two subsets of anti-HLA-DR mAb suggest that anti-id antibodies may play a role in the generation of diversity in the course of a humoral immune response.     

Detection of microsporidia by indirect immunofluorescence antibody test using polyclonal and monoclonal antibodies.

During a screening for monoclonal antibodies (MAbs) to the microsporidian Encephalitozoon hellem, three murine hybridoma cell lines producing strong enzyme-linked immunosorbent assay (ELISA) reactivities were cloned twice, were designated C12, E9, and E11, and were found to secrete MAbs to the immunoglobulin M isotype. On subsequent ELISAs, the three MAbs reacted most strongly to E. hellem, and they reacted somewhat less to Encephalitozoon cuniculi and least to Nosema corneum, two other microsporidian species. The MAbs produced values of absorbance against microsporidia that were at least three times greater than reactivities obtained with control hybridoma supernatants or with uninfected host cell proteins used as antigens. By Western blot immunodetection, the three MAbs detected three E. hellem antigens with relative molecular weights (M(r)s) of 62, 60, and 52 when assayed at the highest supernatant dilutions producing reactivity. At lower dilutions, the MAbs detected additional proteins with M(r)s of 55 and 53. By using indirect immunofluorescence antibody staining, the MAbs, as well as hyperimmune polyclonal murine antisera raised against E. cuniculi and E. hellem, were able to detect formalin-fixed, tissue culture-derived E. cuniculi and E. hellem and two other human microsporidia, Enterocytozoon bieneusi and Septata intestinalis, in formalin-fixed stool and urine, respectively. E. bieneusi, however, stained more intensely with the polyclonal antisera than with the MAbs. Neither the MAbs nor the hyperimmune murine polyclonal antibodies detected Cryptosporidium, Giardia, Trichomonas, or Isospora spp. At higher concentrations, the polyclonal antisera did stain N. corneum and yeast cells. The background staining could be absorbed with Candida albicans. These results demonstrate that polyclonal antisera to E. cuniculi and E. hellem, as well as MAbs raised against E. hellem, can be used for indirect immunofluorescence antibody staining to detect several species of microsporidia known to cause opportunistic infections in AIDS patients.     

Functional analysis of pneumolysin by use of monoclonal antibodies.

We have produced a panel of monoclonal antibodies to pneumolysin, the membrane-damaging toxin from Streptococcus pneumoniae. We have used these antibodies to identify three regions of the toxin sequence that are involved in the lytic mechanism of this toxin. Two of these sites probably form the cell binding site of this toxin. Antibodies to the third site inhibit the lytic action of this toxin but not the binding of this toxin to cells. This site is engaged in the oligomerization process involved in the formation of pores in cell membranes. Two of these epitopes are also present in the related toxin perfringolysin O.     

Investigation of interaction between two neutralizing monoclonal antibodies and SARS virus using biosensor based on imaging ellipsometry

Two neutralizing human scFv, b1 and h12 were identified initially using ELISA,employing highly purified virus as the coating antigen. The biosensor technique based on imaging ellipsometry was employed directly to detect two neutralizing monoclonal antibodies and serial serum samples from 10 SARS patients and 12 volunteers who had not SARS. Further, the kinetic process of interaction between the antibodies and SARS-CoV was studied using the real-time function of the biosensor. The biosensor is consistent with ELISA that the antibody h12 showed a higher affinity in encountering the virus than antibody b1. The affinity of antibody b1 and antibody h12 was 9.5 × 10⁶ M⁻¹ and 1.36 × 10⁷ M^{− 1}, respectively. As a label free method, the biosensor based on imaging ellipsometry proved to be a more competent mechanism for measuring serum samples from SARS patients and the affinity between these antibodies and the SARS coronavirus.     

肝癌单抗重链可变区基因的克隆及序列测定

从分泌与肝细胞肝癌特异性强并具有良好人体内导向作用的鼠源性单克隆抗体的杂交瘤细胞ＨＡｂ２５中提取ＲＮＡ，逆转录成ｃＤＮＡ，用合成的寡核甘酸引物从中扩增出抗体重链可变区基因。将此基因克隆到ｐＵＣ１９质粒中，测定了此可变区基因的全序列。经计算机分析，结果表明基因长度为３６０ｂｐ，编码１２０个氨基酸，是具有功能性的抗体可变区基因，在核苷酸序列上与小鼠重链ＶＨ１８６．２家族同源性最高，属免疫球蛋白重链可变区基因９个家族中的第三族     

Binding properties of monoclonal anti-IgG antibodies: analysis of binding curves in monoclonal antibody systems

The binding properties of an immune complex-forming system comprising human IgG and mouse monoclonal antibodies against human IgG have been studied. A refined binding assay has been applied directly on ascitic fluid containing monoclonal antibody. Complete sets of binding data of a series of different monoclonal antibodies were collected and analysed by various graphical and statistical methods. Special attention was given to methods which allow determination of specific monoclonal antibody concentration as well as antibody affinity. It was found that the formation of genuine antigen: antibody complexes per se gives rise to deviations from expected linearity in commonly used binding equations. Good correlation was found between the antibody concentrations obtained by various graphical approaches, whereas the size of the association constant seemed to depend on the method in use. The binding pattern was found to be dependent on the concentration of antibody. Most reliable parameters were obtained if the product of the antibody concentration and the association constant was below 10.     

Specificity analysis of monoclonal anti-DNA antibodies.

The specificity of a panel of murine monoclonal anti-DNA antibodies for DNA antigenic determinants was evaluated by testing their relative binding to various animal and bacterial DNAs. The antibody panel consisted of six monoclonal anti-DNAs of MRL-lpr/lpr and B6-lpr/lpr origin, while the antigens tested were calf thymus (CT), salmon testes (ST), E. coli (EC) and Micrococcus (MC) DNA. While all antibodies bound to CT, ST, and EC DNA to a similar extent by direct ELISA, only one showed an equivalent level of interaction with MC DNA. The relationship of antigenic sites recognized by the antibodies was evaluated further by competition ELISA, assessing the ability of the anti-DNAs to block the interaction of a biotinylated anti-DNA with solid-phase DNA antigen. For each of the DNAs tested, two patterns of DNA interaction could be distinguished on the basis of the relative inhibitory activity of the different monoclonals. These results suggest that anti-DNA antibodies can be characterized using naturally occurring DNAs, with the observed patterns of binding suggesting recognition of unique antigenic sites, some of which are discrete and non-overlapping.     

Measurement of IgG-blocking antibodies by ELISA using monoclonal antibody and fluorogenic substrate

An enzyme-linked immunosorbent assay (ELISA) using mouse monoclonal antihuman gamma-chain antibody and a fluorogenic substrate has been developed for quantitation of IgG-blocking antibodies in human serum. Generation of fluorescent product was linear with time to 60 min. Using optimal conditions the ELISA was sensitive to less than 1 ng/ml of specific IgG to short ragweed pollen. The assay demonstrated consistently parallel dilution curves with 51 sera (mean interdilutional coefficient of variation = 8.8%). Reproducibility was determined by constructing precision profiles for intra and interassay variation for the entire working range of the assay. Intraassay CVs ranged from a mean of 13% at threshold to less than 5% at higher antibody concentration. Interassay reproducibility similarly ranged from 18 to 10%. In this assay the effect of serum dilution on nonspecific binding was minimal and specific binding of 4-10 ng IgG antibody to the antigen-adsorbed wells was largely complete (75.8 +/- 4.8%) and highly specific (greater than 98%). This application of ELISA for ragweed IgG antibody measurement has performance specifications equal or superior to previously developed radioimmunoassay and ELISA systems.     

Discrimination by rabbit anti-idiotypic antibodies of two murine IgM monoclonal antibodies directed against lipid A.

Two murine IgM monoclonal antibodies (MAs) directed against the lipid A portion of bacterial lipopolysaccharide (LPS) were compared in their binding to Re LPS and lipid A and their idiotypic make-up with rabbit anti-idiotypic sera. Horseradish peroxidase (HRPO)-labelled MAs 8-2 and 26-20 bound equally well to Re LPS. The binding of HRPO-labelled MA 8-2 to synthetic lipid A was low compared to the relatively strong binding of labelled 26-20. The MAs proved to be competitive in a competition binding assay (CBA) with Re LPS as coating antigen. Rabbit immune sera were raised against individual MAs. Anti-idiotypic antibodies (anti-id Abs) were detected with two sensitive enzyme immunoassays (EIA): a solid-phase EIA and an inhibition EIA. The rabbit antisera proved to be idiotype specific, indicating that both MAs recognize separate epitopes. We expect that anti-id Abs will prove to be of value for the differentiation of panels of LPS specific MAs.     

Importance of complement source in bactericidal activity of human antibody and murine monoclonal antibody to meningococcal group B polysaccharide.

The bactericidal activity of human antibody and murine monoclonal antibody to meningococcal group B polysaccharide was investigated as a function of the complement source. The immunoglobulin M murine monoclonal antibody 2-2-B was shown by several different methods to be highly specific for meningococcal group B and Escherichia coli K1 capsular polysaccharides. It had strong bactericidal activity in conjunction with either rabbit or human complement, but gave a higher titer with rabbit complement. A strong prozone was observed in each case. Human postvaccination antibody to meningococcal group B polysaccharide was strongly bactericidal with rabbit complement, but had little or no bactericidal activity in conjunction with human complement. Antibodies in adult normal human sera that were bactericidal with rabbit complement were also found to be predominantly directed against the meningococcal group B capsular polysaccharide. Human antibodies that were bactericidal with human complement appeared to be primarily directed against noncapsular antigens.     

Structural analysis of the CD69 early activation antigen by two monoclonal antibodies directed to different epitopes.

The biochemical structure of CD69 early activation antigen has been characterized by means of two newly isolated mAb, namely C1.18 and E16.5. Upon analysis by SDS-PAGE, C1.18-reactive molecules immunoprecipitated from 125I-surface labeled PMA activated PBL consisted of a 32 + 32 kD dimer, a 32 + 26 kD dimer, a 26 + 26 kD dimer and a 21 + 21 kD dimer. E16.5-reactive molecules consisted of a 26 + 26 kD dimer and a 21 + 21 kD dimer. Cross absorption experiments showed that E16.5 mAb reacts with an epitope of the CD69 molecule distinct from the one recognized by C1.18 mAb and present only on a subpopulation of the CD69 molecular pool. The patterns of migration of C1.18- and E16.5-reactive molecules in two-dimensional gel-electrophoresis, under reducing conditions before and after treatment with Endoglycosidase F enzyme suggest that the two mAb recognize the same glycoprotein structure, but in two distinct glycosylation forms, both expressed on the cell surface membrane. Finally, p32, p26 and p21 of CD69 complex obtained from three distinct normal donors did not show appreciable structural polymorphism, by two-dimensional peptide mapping, not only among single subunits within the same individual, but also among homologous subunits in distinct individuals. Further, it was found that CD69 complex is expressed at the cell surface of resting PBL, although at a very reduced level in comparison to PMA activated cells. C1.18 and E16.5 mAb induced comparable cell proliferation and IL-2 production in PBL in the presence of PMA. C1.18 mAb increased intracellular free calcium concn in PMA activated PBL after cross-linking with goat anti mouse Ig, while the effect induced by E16.5 mAb after cross-linking was consistently lower. Finally, it was found that Sepharose-linked C1.18 mAb, in the presence of rIL-2 or PMA, did not induce TNF release from 6 NK cell clones.     

Microneme antigens of Eimeria bovis recognized by two monoclonal antibodies

Two IgG1 monoclonal antibodies (mAbs 8-23F9 and 9-21G9) were developed after immunization of mice with homogenates of Eimeria bovis first-generation merozoites. Both mAbs reacted with antigens in the apical two-thirds of the parasites and immune electron microscopy determined the micronemes as targets. When tested by immunoblotting, mAb 8-23F9 failed to react with antigens separated under reducing conditions; under nonreducing conditions it recognized two components of >200 kDa. mAb 9-21G9 bound to antigens of 135 and 180 kDa after electrophoresis under reducing conditions and to a series of components when separated without reduction. The epitope of mAb 8-23F9 was destroyed by treatment of the antigen with endoglycosidase H and removal of phosphocholine (PC) by phospholipase C. Since mAb 8-23F9 does not recognize cytidine-linked PC, the data suggest that PC in combination with N-linked sugars and/or N-glycans is part of its epitope. In the case of mAb 9-21G9, endoglycosidase H did not alter the epitope. When E. bovis merozoite antigen was treated with phospholipase C the number of mAb 9-21G9-reactive constituents increased, suggesting that PC may otherwise mask the epitope. mAb 8-23F9 also bound to the apical area and the surface of E. bovis sporozoites and recognized a >200-kDa sporozoite component. When sporozoites invaded Vero cells in vitro, epitope-bearing components were released onto the host cell surface and became part of the early parasitophorous vacuole wall. At day 5 the binding of the mAb was again confined to the intracellular parasite. mAb 9-21G9 did not react with sporozoites but recognized the apical area of intra-cellular trophozoites on day 5 after invasion of host cells in vitro. When testing was done against a variety of other Apicomplexa in various assays, the only cross-reaction observed occurred with mAb 8-23F9, which bound to a conformationally determined 180-kDa component of Toxoplasma gondii cystozoites. Received: 20 November 1998 / Accepted: 18 January 1999     

Dendritic cells of the mouse recognized by two monoclonal antibodies

A new monoclonal antibody, MIDC-8, is described which shows a comparable tissue distribution as the recently described NLDC-145 antibody. It recognizes interdigitating cells, veiled cells and Langerhans cells in lymphoid organs and the skin of the mouse. In contrast to NLDC-145 it recognizes a cytoplasmic component of these cell types. Its distribution is more restricted to the T cell-dependent areas than NLDC-145. Isolation of dendritic cells from lymph nodes, spleen and thymus revealed that both antibodies react with the in vitro isolated dendritic cells. The results show that these antibodies can be used to study dendritic cells in vitro and emphasize the relationship between the in vivo interdigitating cells of the T cell areas and the in vitro isolated dendritic cells.