Regulation of the antibody response to sheep erythrocytes by monoclonal IgG antibodies

Monoclonal antibodies directed against sheep erythrocytes of the isotypes IgG1, IgG2b and IgG2a were used to analyze the specificity of antibody-induced suppression of the immune response. It was first shown that all monoclonals reacted against different antigenic determinants and they all suppressed the immune response to sheep erythrocytes when given shortly after the antigen to more than 50% as compared to 90-96% inhibition obtained with a polyclonal antiserum. Increasing the doses of monoclonals did not increase suppression. However, two different monoclonals administered together caused an additive, but not a synergistic inhibitory effect. No enhancement of the immune response was observed with any of the Ig classes tested. These findings show that four different antigenic determinants on sheep erythrocytes induced the synthesis of corresponding antibodies, with little or no signs of a dominant determinant. Passively administered monoclonal antibodies, even at supraoptimal doses, never suppressed the immune response to the same extent as a polyclonal antiserum, suggesting that each monoclonal only suppressed the synthesis of the corresponding antibody and did not affect antibody synthesis to other determinants.     

Performance of multistep immunochemical reactions by counterflow isotachophoresis on nitrocellulose membranes--II. Epitope analysis of monoclonal antibodies

The method for comparison of epitope specificity of different monoclonal antibodies to one antigen, using a panel of monoclonal antibodies to mouse and human alpha-fetoprotein is described. The method used exploits the special properties of electroendosmotic flow in nitrocellulose membranes under the conditions of anionic isotachophoresis. Electroendosmosis allows successive transfer of several immunoreagents to the dots of monoclonals previously bound to the nitrocellulose membrane. The inhibition of antigen binding to monoclonal dot, if the antigen is mixed with excess of another monoclonal antibody, demonstrates that both monoclonals are directed to the same epitope, and vice versa. The method requires neither purified monoclonals nor antigens, or radio labelling, and is performed semi-automatically. It was shown that each monoclonal antibody to mouse and human alpha-fetoprotein had its own epitope specificity.     

Murine monoclonal antibodies specific for virulent Treponema pallidum (Nichols).

Murine anti-Treponema pallidum (Nichols) lymphocyte hybridoma cell lines secreting monoclonal antibodies against a variety of treponemal antigens have been generated. Hybridomas isolated were of three major types: those that were directed specifically against T. pallidum antigens, those that were directed against treponemal group antigens (as evidenced by their cross-reactivity with T. phagedenis biotype Reiter antigens), and those that cross-reacted with both treponemal as well as rabbit host testicular tissue antigens. The majority (31 of 39 clones) of these anti-T. pallidum hybridomas, which produced monoclonal antibodies of mouse isotypes immunoglobulin G1 (IgG1), IgG2a, IgG2b, IgG3 or IgM, were directed specifically against T. pallidum and not other treponemal or rabbit antigens tested by radioimmunoassay. Four of these T. pallidum-specific hybridomas secreted monoclonal antibodies with greater binding affinity for "aged" rather than freshly isolated intact T. pallidum cells, suggesting a possible specificity for "unmasked" surface antigens of T. pallidum. Six anti-T. pallidum hybridomas produced complement-fixing monoclonal antibodies (IgG2a, IgG2b, or IgM) that were capable of immobilizing virulent treponemes in the T. pallidum immobilization (TPI) test; these may represent biologically active monoclonal antibodies against treponemal surface antigens. Three other hybridomas secreted monoclonal antibodies which bound to both T. pallidum and T. phagedenis biotype Reiter antigens, thus demonstrating a possible specificity for treponemal group antigens. Five hybridoma cell lines were also isolated which produced IgM monoclonal antibodies that cross-reacted with all treponemal and rabbit host testicular tissue antigens employed in the radioimmunoassays. This report describes the construction and characteristics of these hybridoma cell lines. The potential applications of the anti-T. pallidum monoclonal antibodies are discussed.     

Isotype distribution and specificity of the antibody response to primary Moloney murine sarcoma virus infection in BALB/c mice

The development and isotype distribution of Moloney murine leukemia virus (M-MuLV)-specific serum antibodies following primary inoculation with Moloney murine sarcoma/leukemia virus (M-MuSV/M-MuLV) in adult BALB/c mice have been investigated using an enzyme-linked immunosorbent assay (ELISA). The primary antibody responses to M-MuSV/M-MuLV consisted of the IgM, IgG2a, IgG2b, and IgG3 isotypes; no M-MuLV-specific serum IgG1 or IgA antibodies were detected. The detectable antibody response was biphasic, with an early peak of virus-specific titers seen between 10 and 15 days after inoculation and a second peak seen in regressor sera. Pooled regressor sera contained IgM, IgG2a, and IgG2b antibodies which bound to M-MuLV-expressing lymphoma cells. Immunoelectron microscopy with regressor sera showed IgG bound both to infected cell surfaces and to mature viral particles, while IgM bound only to infected cell surfaces. These findings were supported by immunoprecipitation analyses which demonstrated binding of the M-MuLV-specific antibodies to both virion-associated and cell-associated antigens encoded by the gag and env genes.     

Antibody mediated lysis of hapten-conjugated target cells by macrophages and by complement: the influence of IgG subclass, antibody and hapten density.

Lysis of 51Cr-labeled TNP-SRBC sensitised by rat IgG1 or IgG2a type antibodies by homologous, paraffin oil-elicited peritoneal macrophages (ADCC) or by homologous complement was studied. IgG2a was found to be markedly more efficient in mediating both ADCC and complement dependent lysis compared to IgG1. Inhibition of the ADCC pointed to the involvement of separate but partially overlapping interaction sites for the two isotypes. We suggest that FcRII type receptors play a favoured role in both IgG2a and IgG1 mediated ADCC. The threshold amount of bound antibody required for ADCC was lower than that sufficient for complement dependent lysis regardless on the subclass or on hapten density. The extent of lysis (both ways) was found to depend on hapten density using equal amounts of antibody. The results are interpreted as terms of the possible requirements for association of IgG molecules on the target cell surface.     

Photometric assays for FcRI-dependent binding, phagocytosis, and antibody-dependent cellular cytotoxicity mediated by monomeric IgG gamma 2a in murine peritoneal macrophages

Mouse peritoneal macrophages possess distinct Fc receptors (FcR) for binding the various murine IgG isotypes. FcRI binds monomeric IgG gamma 2a, but not monomeric IgG gamma 2b or IgG gamma 1 with high affinity at 4 degrees C and is sensitive to trypsin degradation. We have assessed the functional consequences of the cytophilic binding at 4 degrees C of monomeric IgG gamma 2a to FcRI of mouse peritoneal macrophages using newly developed photometric microassays for quantification of binding, phagocytosis, and antibody dependent cellular cytotoxicity (ADCC) of post-opsonized sheep red blood cell (SRBC) targets. Dose-dependent binding specificity of monomeric IgG gamma 2a, but not IgG gamma 2b or IgG gamma 1 to FcRI of oil-elicited mouse peritoneal macrophages at 4 degrees C for 2 h was confirmed to display typical saturation kinetics both by the photometric assay and by a cellular enzyme-linked immunosorbent assay (CELISA). Binding of monomeric IgG gamma 2a to macrophage FcRI promoted highly efficient phagocytosis of opsonized SRBC in that most cells that were bound were also rapidly internalized by the phagocytic process during a 1 h incubation at 37 degrees C. Upregulation of FcRI-dependent binding and phagocytosis occurred during 24-48 h in vitro culture of macrophages as shown both by the photometric assays and CELISA. Trypsin treatment of macrophages abrogated FcRI-dependent binding and phagocytosis by monomeric IgG gamma 2a, but had little effect on FcRII-dependent functions. Cytophilic binding of monomeric IgG gamma 2a to FcRI failed to trigger ADCC activation. Thus functional characterization of macrophage FcRI-dependent effector functions confirmed the fidelity of binding specificity of monomeric IgG gamma 2a to a trypsin degradable receptor which mediates highly efficient phagocytosis but fails to initiate the signal for ADCC activation. It appears that passively bound immune monomeric IgG gamma 2a could provide an efficient mechanism by macrophages in vivo for FcRI-dependent immune clearance of soluble or particulate cellular antigens without elicitation of potentially harmful cytolytic factors associated with ADCC activation.     

Comparison of cell-surface human melanoma-associated antigens identified by rabbit and murine antibodies

Melanoma-associated antigens (MAAs) recognized by murine monoclonal and rabbit polyclonal antibodies were compared. Two rabbit antimelanoma sera raised in our laboratory recognized five human cell-surface MAAs with approximate MWs of 75, 95, 120, 150, and 240 kd. These antigens were easily detected by SDS-PAGE of specific immunoprecipitates on a melanoma cell (HM31) which failed to reveal antigens reactive with the panel of murine monoclonal antibodies studied. By contrast, these five antigens could not be detected on another melanoma cell (SK-MEL-28), which was reactive with several of the murine monoclonals. These results suggest that the MAAs recognized by rabbit and murine antibodies are different and imply that the antigens which are immunogeneic in man may not necessarily be immunogeneic in mice.     

Lack of evidence for IgM-induced ADCC: studies with monoclonal and polyclonal antibodies.

Different kinds of IgM antibodies were tested for their activity in antibody-dependent cellular cytotoxicity (ADCC): firstly an anti-benzylpenicilloyl (BPO) IgM antibody from immune rabbit serum purified by affinity, ion exchange, and molecular-sieving chromatography, secondly two monoclonal rat anti-BPO IgM antibodies and thirdly a human antidextran antibody prepared from a patient showing restriction of anti-dextran antibodies to the IgM class. Human lymphocytes or purified monocytes served as effector cells. While the two monoclonal rat and the human IgM antibodies showed no ADCC-mediating capacity, ADCC was induced by the rabbit anti-BPO IgM antibody when high antibody concentrations were used. This activity was abolished by further purification using an anti-rabbit IgG (Fc) immunosorbent. The initially observed activity was shown to be likely due to traces of aggregated anti-BPO IgG, which cannot be detected by the methods commonly used. Preincubation of lymphocytes for 24 hr increased the number of EA (IgM)] rosette forming cells but failed to induce IgM-mediated ADCC. Furthermore, evidence for amplification of low-dose IgG-ADCC by IgM could not be found.     

The half-lives of serum immunoglobulins in adult mice

We determined the half-lives of several sets of murine monoclonal antibodies spanning all immunoglobulin isotypes in the serum. The antibodies in each set possess the same V region. With this approach, the differences in half-life observed between the different isotypes are independent of the V region carried by the monoclonal antibodies and therefore must relate to each other in the same way as the half-lives of each class of serum immunoglobulins. The half-life of a monoclonal antibody of the gamma 2a isotype is identical to the average half-life of serum IgG2a as previously determined (6-8 days; P. Vieira and K. Rajewsky, Eur. J. Immunol. 1986. 16:871). Therefore, the half-lives determined with monoclonal antibodies possessing the same V region represent the half-life of the serum immunoglobulins. In this way we calculated the half-life of IgM as 2 days, IgG3 and IgG1 as 6-8 days, IgG2b has a half-life of 4-6 days. IgE has a half-life of 12 h. A polymeric form of IgA was found to be eliminated from the serum with a half-life of 17-22 h.     

A peptide mimic blocks the cross‐reaction of anti‐DNA antibodies with glomerular antigens

Anti‐DNA antibodies play a pivotal role in the pathogenesis of lupus nephritis by cross‐reacting with renal antigens. Previously, we demonstrated that the binding affinity of anti‐DNA antibodies to self‐antigens is isotype‐dependent. Furthermore, significant variability in renal pathogenicity was seen among a panel of anti‐DNA isotypes [derived from a single murine immunoglobulin (Ig)G3 monoclonal antibody, PL9‐11] that share identical variable regions. In this study, we sought to select peptide mimics that effectively inhibit the binding of all murine and human anti‐DNA IgG isotypes to glomerular antigens. The PL9‐11 panel of IgG anti‐DNA antibodies (IgG1, IgG2a, IgG2b and IgG3) was used for screening a 12‐mer phage display library. Binding affinity was determined by surface plasmon resonance. Enzyme‐linked immunosorbent assay (ELISA), flow cytometry and glomerular binding assays were used for the assessment of peptide inhibition of antibody binding to nuclear and kidney antigens. We identified a 12 amino acid peptide (ALWPPNLHAWVP, or ‘ALW’) which binds to all PL9‐11 IgG isotypes. Preincubation with the ALW peptide reduced the binding of the PL9‐11 anti‐DNA antibodies to DNA, laminin, mesangial cells and isolated glomeruli significantly. Furthermore, we confirmed the specificity of the amino acid sequence in the binding of ALW to anti‐DNA antibodies by alanine scanning. Finally, ALW inhibited the binding of murine and human lupus sera to dsDNA and glomeruli significantly. In conclusion, by inhibiting the binding of polyclonal anti‐DNA antibodies to autoantigens in vivo, the ALW peptide (or its derivatives) may potentially be a useful approach to block anti‐DNA antibody binding to renal tissue.     

Specificity and mechanism of immunoglobulin M (IgM)- and IgG-dependent protective immunity to larval Strongyloides stercoralis in mice

Protective immunity in mice to the infective third-stage larvae (L3) of Strongyloides stercoralis was shown to be dependent on immunoglobulin M (IgM), complement activation, and granulocytes. The objectives of the present study were to determine whether IgG was also a protective antibody isotype and to define the specificity and the mechanism by which IgG functions. Purified IgG recovered from mice 3 weeks after a booster immunization with live L3 was shown to transfer high levels of protective immunity to na?ve mice. IgG transferred into mice treated to block complement activation or to eliminate granulocytes failed to kill the challenge larvae. Transfer of immune IgG into IL-5 knockout (KO) mice, which are deficient in eosinophils, resulted in larval attrition, while transfer into FcRgamma KO mice did not result in larval killing. These findings suggest that IgG from mice immunized with live L3 requires complement activation and neutrophils for killing of L3 through an antibody-dependent cellular cytotoxicity (ADCC) mechanism. This is in contrast to the results of investigations using IgM from mice immunized with live L3 and IgG from mice immunized with larval antigens soluble in deoxycholate in which protective immunity was shown to be ADCC independent. Western blot analyses with immune IgM and IgG identified few antigens recognized by all protective antibody isotypes. Results from immunoelectron microscopy demonstrated that the protective antibodies bound to different regions in the L3. It was therefore concluded that while IgM and IgG antibodies are both protective against larval S. stercoralis, they recognize different antigens and utilize different killing mechanisms.     

IgG binding of monoclonal anti-nuclear antibodies from MRL-lpr/lpr mice

To assess the specificity of anti-nuclear antibodies with cross-reactive rheumatoid factor (RF) activity, monoclonal anti-DNA and anti-Sm antibodies from MRL-lpr/lpr mice were tested for binding to a variety of IgG antigens. These antibodies had all been previously identified as binding heterologous IgG. By ELISA, antibodies in this panel all bound BALB/c myeloma proteins representing the different IgG subclasses, indicating broad reactivity with murine IgG as well as heterologous IgG. The determinant recognized by these antibodies was further investigated using the Fab, F(ab')2 and Fc fragments of both human as well as rabbit origin. All antibodies bound well to fragments as well as intact IgG antigens. These antibodies were further analysed by Western blotting, demonstrating that most bound to both heavy and light chains of human origin. Together, these observations suggest that some anti-nuclear antibodies bind a conserved antigenic determinant present widely on immunoglobulin chains. This determinant may represent a common sequence important in immunoglobulin domain structure.     

Monoclonal Antibodies to HLA–DRw Determinants

Two monoclonal antibodies recognizing HLA-DRw (human la) antigens were produced. The DA2 antibody binds a monomorphic determinant, common to all specificities and Genox3.53 antibody binds to a cross-reacting site on the HLA-DRw1,2 and 6 specificities. Both antibodies are IgG1 and show complement dependent cytotoxicity only in the presence of rabbit anti-mouse IgG serum. Specificity of both antibodies for the HLA-DRw molecule was shown by inhibition of antibody binding by preincubation of antibody with detergent solubilized Ia from JY (HLA-DRw4,6) cells and by preincubation of target cells with F(ab')2 fragments of a rabbit anti-la serum. DA2 antibody reacted with all cells of human B cell origin tested and with peripheral blood lymphocytes of several primate species tested. Genox3.53 antibody bound only to human cells expressing HLA-DRw1,2 or 6 antigens, giving a negative reaction with all primates tested. Genox3.53 antibody detected a split in the HLA-DRw6 specificity, showing reduced binding to the Daudi cell (HLA-DRw6) in comparison with binding to several other cell lines typed as HLA-DRw6, under saturating conditions. This low reactivity with Daudi was confirmed by absorption experiments. The ratio of DA2 binding to Genox3.53 binding to homozygous and heterozygous cell lines under saturation conditions was compared. Results suggested that, on some cell lines, DA2 might be reacting with a second population of human Ia antigens in addition to the HLA-DRw antigens. When a mixture of saturating concentrations of DA2 and Genox3.53 antibodies was tested for binding to cells under saturating conditions, the number of counts bound suggested the antibodies could bind simultaneously. Direct binding experiments showed that when each antibody was iodinated, its binding was not inhibited by preincubation with the other antibody, confirming that the DA2 and Genox3.53 determinants are distinct on the Ia molecule.     

High concentrations of therapeutic IgG1 antibodies are needed to compensate for inhibition of antibody-dependent cellular cytotoxicity by excess endogenous immunoglobulin G

A common feature of human IgG1 antibodies used for cancer treatment is that their anti-tumour efficacy requires high serum trough levels and continued therapy for several months. Treatment cycles, thereby, consume several grams of IgG1 translating into significant drug needs and costs. The basis for the low in vivo efficacy, which is in contrast to high in vitro antibody-dependent cellular cytotoxicity (ADCC), is not well understood. Here, we have explored factors contributing to this discrepancy using adecatumumab (MT201), a fully human monoclonal IgG1 against epithelial cell adhesion molecule (Ep-CAM) and trastuzumab (Herceptin), a humanized IgG1 with specificity for the human epithelial growth factor receptor type 2 (HER-2) antigen. We found that physiological levels of human sera strongly inhibited ADCC of both IgG1 antibodies. Effects showed some dependence on the density of Ep-CAM and HER-2 targets, the tumour cell line tested and on effector cell and serum donors. Removal of IgG by affinity chromatography abolished the inhibitory effect of a serum pool. Inhibition of ADCC was fully restored by adding back the IgG fraction or by an equal amount of IgG from a commercial source. We further demonstrate that CD56-positive lymphocytes within human PBMC contributed >90% to ADCC and that normal serum levels of IgG effectively competed for in vitro binding of an IgG1 antibody to low-affinity Fcgamma receptor type III (CD16), as is present on natural killer (NK) cells. Competition of serum IgG for binding of therapeutic IgG1 to NK cell may be one important reason why high antibody doses are required in the clinic for treatment of cancer by an ADCC-based mechanism.     

Regulation of IgG Antibody-dependent Cellular Cytotoxicity in Vitro by IgM Antibodies

IgM antibodies have previously been reported to either inhibit or induce antibody-dependent lymphocyte cytotoxicity (ADCC). Here we show that human lymphocytes lyse bovine erythrocytes (E_b) in the presence of either IgM or IgG anti-E_b from rabbits. Seven out of 20 IgM preparations (Sephadex G-200) were ADCC-active. IgG-dependent ADCC was inhibited by human IgG but not by IgM. In contrast, IgM ADCC was inhibited by both IgG and IgM. The effector cells in IgM ADCC were a subpopulation of lymphocytes with distinct Fc receptors for both IgG and IgM. Most of them also had sheep erythrocyte receptors. Extensive purification of the ADCC-active IgM antibody preparations indicated that very small amounts of contaminating IgG anti-E_b were responsible for ADCC induction. When purified and ADCC-inactive IgM antibodies were mixed with suboptimal concentrations of IgG antibodies, a strong enhancement of ADCC was found. To achieve enhancement, the two antibody isotypes had to be present on the surface of the same target cells, and the IgM effect was not due to the release of soluble ADCC-enhancing factors. Thus, in this system, IgM antibodies are not capable of inducing ADCC on their own. However, they enhance ADCC by improving the contactual interaction between target cells and a special subset of effector cells.     

应用免疫微球技术检测小鼠T、B淋巴细胞表面标志的探讨

本文报道用羧化聚苯乙烯胶乳微球为载体,分别用抗小鼠 Thy1.2单克隆抗体及纯化的抗小鼠 Ig 抗体与其交联制备成免疫微球,应用于检测小鼠 T、B 淋巴细胞,并探讨本实验的各种影响因素,同时用补体依赖性细胞毒实验及间接免疫荧光法作比较,所得结果一致。     

Comparative study of idiotypes on monoclonal antibodies derived from patients with lupus and leprosy and from normal individuals

A collaborative study was performed to compare the expression of a series of idiotypes defined on human anti-DNA and other autoantibodies. Three panels of human monoclonal antibodies were tested: eight derived from patients with systemic lupus erythematosus (SLE); 13 from an individual with lepromatous leprosy; and 38 from normal subjects. The following rabbit anti-idiotype sera were used: one (RId16/6) raised against the lupus-derived monoclonal anti-DNA antibody 16/6, four (RId8E7, RId4G7, RId4D5 and RIdTH9) against leprosy-derived monoclonal antibodies of various specificities, and one (anti-4.6.3) against a normal-derived anti-DNA monoclonal (KIM 4.6). In addition, two other anti-idiotypes were used--one a murine monoclonal (3I), the other a rabbit polyclonal (RIdD)--which had been raised against polyclonal anti-DNA antibodies from lupus serum. Further experiments were performed with immunoabsorbed fractions of RId8E7. Direct-binding and competition assays were used. All of the anti-idiotypes produced different patterns of positivity among the three panels of human monoclonal antibodies, with the exception of RId8E7 and RId4G7, which showed considerable concordance. There was a tendency towards anti-idiotypes being disease- or group-specific: thus anti-4.6.3 failed to bind to any of the lupus or leprosy-derived monoclonals, while RId16/6 and RId8E7 bound most strongly to the lupus- and leprosy-derived antibodies respectively. KIM 4.6 itself was bound only weakly by RId16/6, while 16/6 was not recognized by anti-4.6.3; 16/6 was, however, bound by 3I, while KIM 4.6 was not. 3I bound to several other monoclonals but RIdD, which has been shown to be specific for the anti-DNA fraction of lupus serum, did not bind to any of them. These results indicate that the majority of these anti-idiotype preparations recognize largely separate sets of determinants. The monoclonal antibodies which bind to DNA may be only partly representative of anti-DNA antibodies in the serum of lupus patients.     

Human and murine antibodies to rye grass pollen allergen LolpIV share a common idiotope.

The possibility that a murine monoclonal antibody (mAb 12) to Rye grass pollen allergen LolpIV and LolpIV-specific antibodies in the sera of grass allergic individuals share a common idiotope (Id) was investigated. It was first established that mAb 12 and human IgE antibodies recognized the same (or similar) epitope(s) on LolpIV; i.e. mAb 12 could inhibit, to the extent of 35-60%, the binding of 125I-LolpIV to the human IgE antibodies present in the sera of grass pollen-allergic individuals. Subsequently, a rabbit anti-Id antiserum was produced against mAb 12 and rendered Id-specific by appropriate immune absorptions, and its IgG antibody fraction was isolated (Rb-aId). The specificity of Rb-aId was demonstrated by the fact that the antibodies bound only to mAb 12 and not to any other murine monoclonal antibody tested. Observations that Rb-aId inhibited the binding of 125I-LolpIV to mAb 12 indicated that the Id determinants recognized on mAb 12 were located at or near the antibody-combining sites. The Rb-aId also bound specifically to affinity-purified human anti-LolpIV antibodies isolated from human sera, but not to affinity-purified human anti-tetanus toxoid antibodies. This indicated that the human anti-LolpIV antibodies share a cross-reactive Id. The binding of Rb-aId to human anti-LolpIV antibody could also be inhibited by mAb 12. Therefore, it was concluded that the murine and human antibodies to LolpIV share a cross-reactive idiotope.     

Opsonization of Cryptococcus neoformans by a family of isotype-switch variant antibodies specific for the capsular polysaccharide

A family of immunoglobulin isotype-switch variants was isolated by sib selection from a murine hybridoma which produced an immunoglobulin G subclass 1 (IgG1) antibody specific for the capsular polysaccharide of Cryptococcus neoformans. Antibodies of the IgG1, IgG2a, and IgG2b isotypes had similar serotype specificity patterns in double immunodiffusion assays which used polysaccharides of the four cryptococcal serotypes as antigens. A quantitative difference in the ability of the isotypes to form a precipitate with the polysaccharide was observed in a double immunodiffusion assay and confirmed in a quantitative precipitin assay. The relative precipitating activity of the antibodies was IgG2a greater than IgG1 much greater than IgG2b. Analysis by enzyme-linked immunosorbent assay of the reactivity of the three isotypes with cryptococcal polysaccharide showed identical titers and slopes, suggesting that the variable region of the class-switch antibodies was unaltered. This system allowed us to examine the effect of the Fc portion of the antibody on opsonization of encapsulated cryptococci. Yeast cells were precoated with antibodies of each isotype and incubated with murine macrophages or cultured human monocytes. Antibodies of all three isotypes exhibited a dose-dependent opsonization for phagocytosis by both human and murine phagocytes. The relative opsonic activity of the antibodies was IgG2a greater than IgG1 greater than IgG2b.     

The importance of antibody isotype in HIV-1 virus capture assay and in TZM-bl neutralization

The binding of murine IgM mAbs to five different clades of HIV-1 was examined using a modified ELISA-based virus capture assay. Two murine multispecific IgM mAbs that exhibit both lipid and gp41 epitope specificities, and one murine IgM mAb that exhibits lipid-binding specificity, were utilized. The binding of the IgG and the IgM isotypes of human mAb 2F5 to clades A through AE were also evaluated. The binding of 2F5 to HIV-1 was dependent upon the antibody isotype. Monoclonal IgM antibodies bound significantly lower amounts of HIV-1 than the corresponding IgG isotype. Although murine IgM mAbs bound HIV-1 to varying degrees in the virus capture assay, they failed to neutralize HIV-1 in a TZM-bl pseudovirus assay. In contrast, 2F5-IgM mAb bound certain HIV-1 isolates, and also neutralized them, although not as efficiently as the 2F5-IgG isotype. Studies on the relationship between virus binding and neutralization in a TZM-bl pseudovirus assay indicated that in most cases, mAbs that exhibited neutralization also bound the virus.