Transfer of monoclonal antibodies into mammalian cells by electroporation

A simple rapid and reproducible procedure for transferring monoclonal antibodies into mammalian cells by electroporation is described. Two functionally different monoclonal antibodies (Mab 3F3 and Mab 2B4) specific for asparagine synthetase (EC 6.3.1.1) were used for electroporation into HeLa, HT-5, and L5178Y D10/R (L-asparaginase-resistant) cells. The conditions were optimized so that the viability of the electroporated cells was very high (80-90%), and 90% of the viable cells had antibody incorporated. Electropermeabilized cells were structurally intact, and the high voltage electric pulse had no inhibitory effect on overall cellular DNA and protein synthesis. Incorporated immunoglobulins showed unaltered structural integrity and were functionally active. L5178Y D10/R cells incorporated with an antibody (Mab 3F3) known to be a potent inhibitor of tumor asparagine synthetase showed increased dependence on an exogenous source of asparagine in the culture medium, while the growth of cells incorporated with a control (noninhibitory) antibody (Mab 2B4) remained unaffected. These studies demonstrate that electroporation can be employed successfully for large scale transfer of antibodies into cultured mammalian cells for the study of cellular metabolism.     

An interspecies comparison of placental antibody transfer: New insights into developmental toxicity testing of monoclonal antibodies

There are profound differences in maternofetal transfer of immunoglobulins between species with extensive gestational transfer of maternal immunoglobulins in primates (including humans) via the chorioallantoic placenta as well as in rabbits and guinea pigs via the inverted yolk sac splanchnopleure. In contrast, other neonatal rodents (rats and mice) receive passive immunity predominantly postnatally. This transfer is mediated principally via FcRn receptors. Therapeutic monoclonal antibodies (mAbs) are most commonly of the IgG1 subclass, which is transported most efficiently to the fetus. In all animal species used for testing developmental toxicity, fetal exposure to IgG is very low during organogenesis, but this increases during the latter half of gestation such that the neonate is born with an IgG1 concentration similar to the mother (but not rats and mice). Review of mAb developmental toxicity studies of licensed products reveals Cynomolgus monkey as the species used in the majority of the cases (10 out of 15). Pregnancy outcome data from women gestationally exposed to mAb is limited. In general, the findings are consistent with the expected low exposure during organogenesis. Guinea-pigs and rabbits are potential candidates as “alternatives” to the use of nonhuman primates as the maternofetal transfer in the last part of gestation is at a level similar in humans. Based on the pattern of placental transfer of IgG in humans, study designs that allow detection of both the indirect effects in early gestation plus the effects of direct fetal exposure in mid and late gestation are recommended for developmental toxicity of mAbs. Birth Defects Res (Part B), 86:328–344, 2009. © 2009 Wiley-Liss, Inc.     

Detection of new MHC mutations in mice by skin grafting, tumor transplantation and monoclonal antibodies: a comparison

Two mechanisms of major histocompatibility complex (MHC) mutations have been described in mice; gene conversion and homologous but unequal recombination. However, our knowledge of mutations in MHC is incomplete because studies have been limited almost exclusively to two haplotypes, H-2b and H-2d, while hundreds of haplotypes exist in nature; it has been biased by the use of only one procedure of screening for mutation, skin grafting. We used three procedures to screen for MHC mutations: (1) conventional techniques of skin grafting, (2) syngeneic tumor transplantation and (3) typing with monoclonal anti-MHC antibodies (mAbs) and complement. The faster technique of tumor transplantation detected mutants similar to those discovered by skin grafting technique. Screening with mAbs allowed us to detect both mutants that are capable of rejecting standard skin grafts and those that are silent in skin grafting tests, and which therefore resulted in a higher apparent mutation frequency. Two mutants of the H-2a haplotype were found that carry concomitant class I and class II antigenic alterations. Both MHC mutants silent in skin grafting tests and mutants carrying concomitant class I and class II alterations have never been studied before and are expected to reveal new mechanisms of generating MHC mutations. 1-Ethyl-1-nitrosourea (ENU) failed to induce de novo MHC mutations in male mice in our skin grafting series.     

Analysis of anti-tumor antibodies in mice and rabbits induced by monoclonal anti-idiotope antibodies

Eight different mouse monoclonal anti-idiotope antibodies (mAb2) generated against a mouse monoclonal anti-human melanoma proteoglycan Ag (MPG) antibody (mAb1), MEM136, were tested for their ability to induce anti-MPG responses in mice and rabbits. All Ab2 were idiotypically cross-reactive and combining site-specific as demonstrated by competitive cross-inhibition studies and their ability to inhibit the binding of MEM136 to the melanoma cells, Colo38. However, only two Ab2, IM32 and IM06, were able to induce specific anti-TAA-specific (Ab1') responses in rabbits. When IM32 and IM06 were tested in allogeneic stains of mice for the induction of anti-MPG responses, only IM32 produced an Ab1' response. In mice, the Ab3 response induced by IM32 is idiotypically cross-reactive with its Ab1. Furthermore, the IM32-induced murine Ab3 and MEM136 recognized a similar MPG epitope on the melanoma cells because the Ab3 inhibited the binding of MEM136 to melanoma cells. The Ab3 induced by IM32 and IM06 in rabbits also recognized a similar epitope as the Ab1. In rabbits, the Ab3 response induced by IM32 and IM06 were idiotypically cross-reactive with each other. However, additional studies indicated that the majority of Ab3 induced by IM32 were IM32 Id-specific and lacked IM06 idiotopes. Further experimentation indicated that IM32-induced rabbit Ab3 were biologically active as demonstrated by the ability of the Ab3 to inhibit melanoma cell invasion in a Matrigel assay.     

Electron microscopic mapping of monoclonal antibodies on the tail region of Dictyostelium myosin

The binding sites of five monoclonal antibodies against myosin of Dictyostelium discoideum have been mapped. These antibodies bind to the tail region of the myosin molecule. By rotary shadowing, images of myosin-antibody complexes were obtained in which the mean distance of the midpoint of an antibody molecule from the myosin heads was localized with a precision better than 2 nm (90% confidence limit). Other quantitative data extracted from electron micrographs provided information on the stoichiometry of antibody-myosin interaction. Certain antibodies interacted with myosin molecules only at a ratio of 1:1. Other antibodies formed complexes of two molecules bound to homologous sites on a double-stranded myosin tail. Affinities were estimated and the abilities of different antibodies to cross-connect two myosin molecules were evaluated.     

Incorporation of monoclonal antibodies into cells by osmotic permeabilization. Effect on cellular metabolism

Incorporation of asparagine synthetase-specific monoclonal antibodies into L5178Y D10/R (L-asparaginase-resistant) murine lymphoma cells by osmotic lysis of pinocytic vesicles was used to evaluate the potential of the technique for macromolecular incorporation for metabolic studies. Nonspecific effects of the incorporation procedure included temporary inhibition of protein and DNA synthesis by 80-85% and a transitory loss of membrane integrity. Cells incorporated with an antibody inhibitory to tumor cell asparagine synthetase showed increased dependence upon an exogenous source of asparagine in the culture medium, while cells incorporated with a control antibody were not affected. These studies demonstrated that incorporation of inhibitory monoclonal antibodies into cells can be used to study the short term metabolic role of specific enzymes; however, the metabolic effects induced by the specific macromolecule must be evaluated within the context of the nonspecific effects caused by the osmotic treatment required for incorporation.     

Ontogeny of human hematopoietic cells: analysis utilizing monoclonal antibodies

Lymphoid and myeloid cells isolated from second trimester fetal lymphoid organs were characterized by utilizing a panel of monoclonal antibodies that define human lineage-restricted, differentiation, histocompatibility, and activation antigens. At distinct gestational stages, the appearance of morphologically identifiable lymphoid and myeloid cells paralleled the appearance of cells expressing definable lymphoid and myeloid antigens. The proportion of cells in fetal liver, bone marrow, and spleen that expressed histocompatibility, myeloid, and B cell antigens increased with fetal maturation. In contrast, even the earliest fetal thymuses studied were of a phenotype no different than that seen during later stages of ontogeny. Although the cellular lineage of most fetal hematopoietic cells could be identified by this panel of reagents, a considerable number of fetal liver and bone marrow cells did not express any of these antigens, suggesting the possibility that they might represent early hematopoietic progenitor cells. These studies support the notion that the adult cellular phenotype is the result of both an orderly acquisition of differentiation antigens and the migration of these primitive cellular populations to specific fetal organs. Identification of hematopoietic progenitors in fetal tissues may facilitate the identification and isolation of early lymphoid and myeloid progenitor cells in adults.     

Specificity of monoclonal antibodies against human thyroglobulin; comparison with autoimmune antibodies.

Ten monoclonal antibodies (mAb) directed against human thyroglobulin (hTgb) were produced, purified and characterized. The mAb avidity for hTgb ranged from 10(-10) to 10(-6) M. The species specificity of the mAb was as follows: eight mAb reacted with monkey Tgb, three with dog Tgb and one with pig Tgb; none with bovine and ovine Tgb. The binding of mAb to hTgb was not significantly inhibited in the presence of Tgb carbohydrate moieties, tyrosine, iodotyrosines and iodothyronines. The topology of the antigenic determinants recognized by the 10 mAb on hTgb was explored by inhibition of Tgb binding of radiolabeled mAb by the other antibodies. Six distinct clusters of reactivity were described. Localization of the antigenic determinants recognized by mAb on hTgb was attempted using tryptic fragments of hTgb to inhibit the binding of mAb to hTgb. The inhibitory effect of hydrolysis products was different for each mAb but exhibited partial analogies between mAb of the same cluster of reactivity. Anti-hTgb autoimmune antibodies (aAb) purified from sera of Graves patients cross-reacted essentially with mAb of one out of the six clusters. These results demonstrate that the large number of antigenic determinants presented by the hTgb are not disseminated on the molecule but are clustered in antigenic regions. Furthermore, from the six antigenic regions evidenced in this paper, only one is involved in autoimmune antibody production in Grave's disease.     

Production and some characteristics of monoclonal antibodies against beet necrotic yellow vein virus

Four rat monoclonal antibodies (MAbs) specific for beet necrotic yellow vein virus (BNYVV) were produced. In indirect ELISA, all four MAbs reacted strongly with BNYVV infected plant leaf extracts (19 isolates from eight countries) but they did not react with beet soil-borne virus (BSBV), an unnamed rod-shaped soil-borne beet virus isolate (86 - 109) from Sweden or barley stripe mosaic virus (BSMV). However, two of the MAbs, MAFF 6 and MAFF 7 did not detect BNYVV in ELISA of infected sugar beet roots whereas MAbs MAFF 8 and MAFF 9 did detect virus in root extracts. In electro-blot immunoassay (EBIA), MAFF 6 and MAFF 7 readily detected BNYVV coat protein from leaf extracts whereas MAFF 8 and MAFF 9 reacted only weakly. None of the MAbs reacted with BSBV, 86 - 109, BSMV or plum pox virus in EBIA. MAFF 6 coated BNYVV particles which were trapped from infected leaf or root sap on to electron microscope grids by polyclonal antibodies. MAFF 6 was partially purified from tissue culture supernatant fluid by cation exchange chromatography and the preparation used to coat microtitre plates and successfully trap BNYVV in ELISA of leaf sap extracts.     

Production of antigen-specific human monoclonal antibodies: comparison of mice carrying IgH/kappa or IgH/kappa/lambda transloci

Here we compare human monoclonal antibody (MAb) production from mouse strains that carry disruptions of their endogenous mouse IgH/IgK loci and harbor human IgM + Igkappa(BABkappa) or human IgM + Igkappa + IgA transloci (BABkappa,lambda). We found that whereas both strains proved effective for the isolation of antigen-specific IgM antibodies, many of the IgM MAbs elicited from BABkappa comprise human mu chains that are associated with mouse lambda chains. In contrast, BABkappa,lambda mice gave rise to fully functional, polymeric human IgM antibodies comprising both human IgH and human IgL chains. Therefore, the inclusion of a human Iglambda translocus (in addition to the human IgH + Igkappa transloci) not only diminishes problems of endogenous mouse Iglambda expression but also provides a strain of mice that yields fully human MAbs to a wide range of antigens, as witnessed by the isolation of MAbs to human blood cells, tumor cell lines, and an immunoglobulin idiotype.     

Melittin-specific monoclonal and polyclonal IgE and IgG1 antibodies from mice

Melittin, a bee venom peptide consisting of 26 amino acid residues, elicited high IgG and IgE antibody responses in mice of BALB/c and CAF1 strains, but not in mice of A/J, AKR, and C57BL/6 strains. Greater than 80% of the melittin-specific antibodies in sera of responder mice were found to bind the hydrophilic carboxyl-terminal heptapeptide of melittin. Three melittin-specific monoclonal antibodies were obtained from responder mice by the hybridoma technique. Two are of the IgG1 isotype and one is of the IgE isotype. One monoclonal antibody of the IgG1 isotype binds the carboxyl-terminal heptapeptide of melittin, while the other two monoclonal antibodies do not. However, competitive binding studies suggest that all three monoclonal Ig bind at the same, or adjacent, site of melittin. These findings, together with the known amphiphilic property of melittin, suggest that the immunogenicity of this peptide is a consequence of its binding to cell surface phospholipids.     

Passive release of monoclonal antibodies from hybridoma cells

Evidence is presented for the passive release of monoclonal antibodies (MCAB) from hybridoma cells grown in either batch or continuous-flow culture. This release is promoted at room temperature. Passively released MCAB is indistinguishable from that released by actively growing cells, as judged by SDS-polyacrylamide gel electrophoresis. The significance of these observations in relation to the continuous culture of hybridoma cells is discussed.Maximum MCAB content of TB/C3 hybridoma cells is about 55pg per cell, any additional MCAB produced is secreted.Abbreviations MCAB monoclonal antibodies - PBS phosphate buffered saline - RT room temperature - SDS sodium dodecyl sulphate     

Nematospiroides dubius: passive transfer of protective immunity to mice with monoclonal antibodies

Nine hybridoma cell lines secreting monoclonal antibodies specific for Nematospiroides dubius were produced by fusion of the mouse myeloma cell line NS-1 to either spleen cells or mesenteric lymph node cells from mice repeatedly infected with N. dubius. Seven of the antibodies were identified as IgM and two as IgG1. Each monoclonal antibody bound to polypeptide epitopes on both infective larvae (L3) and adult worms. However, five antibodies bound preferentially to L3 and three to adult worms. All nine antibodies reacted with high molecular weight protein antigens. Passive protective immunity in Balb/c mice was demonstrated with monoclonal antibodies Nd2 and Nd3 in ascites fluid which stunted both male and female worms and reduced parasite fecundity.     

Characterization of monoclonal anti-fluorescein antibodies from autoimmune New Zealand mice

Previous studies of murine IgM hybridoma protein 18-2-3, derived from an (NZB/NZW)F1 secondary response to fluorescein (FL) presented on T-dependent carrier, demonstrated a high binding affinity for FL (KA = 2.9 X 10(10) M-1) and cryoprecipitation, which could be abrogated upon FL binding. Based on these unusual properties and their possible association with defective immune regulation in lupus-prone mice, further studies were carried out to evaluate the basis of 18-2-3 cryoprecipitation, expression of characteristics related to the 18-2-3 clonotype, and structure/function aspects of additional homogeneous IgM and IgG antibodies of similar origin and specificity. Solubility experiments in which the effect of ionic strength on macroscopic aggregation was measured indicated that 18-2-3 intrinsically possessed both cryoglobulin and euglobulin properties in the absence of auxiliary gamma-globulin components. Rates of hapten fluorescence quenching by 18-2-3 were limited by factors other than diffusion and were dependent on solution temperature and ionic strength. Thirty-seven additional IgM and IgG monoclonal antibodies were shown to possess normal low-temperature solubility and hapten fluorescence-quenching properties, suggesting that 18-2-3 was derived from a relatively rare B cell progenitor. Collective results from FL binding and spectrotype analyses indicated that the majority of proteins were diverse with respect to variable region structure and binding mechanisms but unusually restricted in binding affinities (KA less than 5 X 10(6) M-1). Relative subclass frequencies for 30 monoclonal IgG proteins (IgG1 greater than IgG2b greater than IgG2a greater than IgG3) were consistent with polyclonal IgG subclass expression in normal mice in response to T-dependent immunogen.     

Plasmodium yoelii: comparison of indirect immunofluorescence and radioimmunoassay for detecting monoclonal antibodies to malaria

The techniques of indirect immunofluorescence (IFA) and radioimmunoassay (RIA) were compared for efficiency in detecting antimalarial monoclonal antibodies in culture supernatants following lymphocyte hybridization. Culture supernatants from 479 wells were examined. Approximately 9% of the wells were found to contain antibodies to Plasmodium yoelii by IFA and 13% by RIA analysis. However, only 4.6% of the wells were positive by both IFA and RIA techniques. In general, stage-specific hybrids were more easily detected in IFA than RIA tests, whereas monoclonal antibodies binding to non-stage-specific antigens were more readily detected by RIA than IFA analysis. Selected monoclonal antibodies of the IgG and IgM classes were purified by protein A and molecular-weight-exclusion chromatography, respectively. The minimal amount of monoclonal antibody required to give a positive IFA reaction ranged from 1.6 to 100 μg antibody/ml (0.03-2 μg antibody/test), but only 0.1 to 100/μ.g antibody/ml (1 ng-2 μg antibody/test) was needed to produce a positive RIA reaction. Use of an isotype-specific RIA for screening for antimalarial antibodies resulted in the detection of additional hybridomas missed by standard IFA and RIA tests. Thus, it is important to use several serologic methods if maximal efficiency in detecting antimalarial monoclonal antibodies is to be achieved.     

Isolation and characterization of monoclonal antibodies reactive with endothelial cells

Monoclonal antibodies were generated to antigens on cultured human umbilical vein endothelial cells. Spleen cells from BALB/c mice, immunized with low passage cultures of human umbilical vein endothelial cells, were fused with the non-secretory myeloma line, P3 x 63Ag 8.653. Hybridoma supernatants were screened for the desired immunological reactivity using ELISA binding assays. Hybridomas secreting antibodies reacting with the immunizing endothelial cells, but not with peripheral blood mononuclear cells, were cloned by limiting dilution and three stable clones were chosen for study. Further testing by ELISA revealed that each antibody displayed a unique pattern of reactivity. One antibody, 14E5, reacted with the macrophage-like cell line DHL-2, cultured macrophages derived from peripheral blood monocytes, and macrophages derived from malignant effusions. The antibody failed to react with fibroblasts or bovine endothelial cells. The second antibody, 12C6, reacted with human and primate fibroblasts and endothelial cells derived from bovine arteries, but not with mature macrophages. The third clone, 10B9, reacted only with the immunizing endothelial cells and the immature-macrophage line U-937. All three antibodies failed to react with long-term human B or T lymphoblastoid cell lines, leukemic cell lines, or murine macrophage lines. None of the antibodies reacted with a battery of human epithelial derived cell lines or primary cultures of human epithelial cells. Indirect immunofluorescence assays revealed that the antigens were expressed on the cell surface. These antibodies should prove useful as differentiation markers of human endothelial cells and in studies of endothelial cell function.