Human monoclonal anti-nuclear antibodies produced by human-mouse heterohybridomas

To obtain human monoclonal anticentromere antibodies, mouse myelomas were fused with unfractionated mononuclear cells from the peripheral blood of a patient diagnosed as having the CREST variant of scleroderma: with only anticentromere antibodies. After a single fusion an heterohybridoma secreting a human antibody specific for nuclear antigens, as detected by indirect immunofluorescence staining, was isolated. The monoclonal antibody secreted by the clone was of the human IgM class. Indirect immunofluorescence staining of the antibody on HEp-2 cells showed multiple nuclear dots or a discrete speckled pattern resembling that of an anticentromere antibody. Immunoblot analysis showed antibody binding to a 33 kD antigen derived from the nuclear protein fraction. Enzyme-immunoassay results clearly showed that the antibody reacted with the chromosomal protein fraction and not calf thymus DNA.     

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.     

Mouse monoclonal antibodies against rat major histocompatibility antigens. Two Ia antigens and expression of Ia and class I antigens in rat thymus

A rat Ia (RT-1B) antigen (called Ia-A) equivalent to the mouse I-A product has been defined with mouse monoclonal antibodies (W. R. McMaster and A. F. Williams, Eur. J. Immunol. 1979. 9: 426). To identify other Ia antigens mouse monoclonal antibodies were raised against rat spleen glycoproteins depleted of the Ia-A antigen. An IgG antibody (called MRC OX17) was obtained and used to purify a molecule which had a similar structure to the Ia-A antigen and reacted with anti-Ia alloantibodies. There was no cross-reaction between the two Ia glycoproteins in assays with mouse monoclonal antibodies, alloantibodies or rabbit antibodies. In one alloantiserum almost all the detectable anti-Ia antibodies reacted with a mixture of the two Ia glycoproteins. The MRC OX17 antibody did not bind to mouse cells, but rabbit antibodies to the pure rat glycoprotein cross-reacted and recognized determinants mapping to the mouse I-E region. In the thymus the rat Ia-E antigen was on cortical epithelial and medullary reticular cells. An IgG monoclonal antibody (MRC OX18) to isotypic determinants of rat histocompatibility RT-1A antigens was also produced and used to analyze these antigens on thymus cells. The heavily labeled thymocytes were those with characteristics of mature T lymphocytes. Cortical epithelial cells and medullary dendritic-like cells were also RT-1A positive.     

The production of human monoclonal anti-MMTV antibodies by in vitro immunization with anti-idiotypic antibodies.

We have previously reported the production of a series of human monoclonal antibodies reacting with mouse mammary tumor viral antigens as well as the human counterpart HuMTV antigen. Against two of these antibodies (B11 and 4.6/6), anti-idiotypic antibodies were generated, which appeared to be the internal image of viral antigen. Vaccination with the anti-idiotypic antibodies induced in mice humoral and cellular immunity against both MMTV and HuMTV. The current study describes a novel method to produce human anti-MMTV antibodies derived monoclonal antibodies. Normal peripheral blood lymphocytes (PBLs) were immunized in vitro in the presence of IL-2, with rabbit anti-B11 antibodies bound to silica beads. Following in vitro immunization, the lymphocytes were fused with the UC-792-6 human lymphoblastoid cell line. Four human hybridomas were found to secrete human monoclonal antibodies which bound to MMTV and HuMTV. Three of the secreted antibodies were of the IgG class and one of the IgM class. The specificity of binding was confirmed by direct and competitive ELISA assays and by radioimmunoprecipitation. Our study demonstrates the ability to generate human monoclonal anti-viral antibodies by in vitro immunization, employing "internal image" anti-idiotypic antibodies. The method can be used to generate human antibodies, especially against pathogenic agents or ill-defined antigens.     

Detection of specific immunoglobulin M antibodies to cytomegalovirus by using monoclonal antibody to immunoglobulin M in an indirect immunofluorescence assay.

The detection of immunoglobulin M (IgM) antibodies to cytomegalovirus-induced late antigens by an indirect immunofluorescence assay was improved by the use of monoclonal antibodies to human IgM. Nonspecific background fluorescence was absent, facilitating the reading of the slides and the detection of a specific fluorescence reaction in sera with low levels of specific IgM. Moreover, the indirect immunofluorescence assay with monoclonal antibodies to IgM proved more sensitive than the indirect immunofluorescence assay with polyclonal antibodies to IgM. The absorption of human sera on staphylococcal protein A avoided false reactions due to the presence of rheumatoid factor and high levels of specific IgG in test sera.     

Human-human hybridoma producing monoclonal antibodies against colorectal cancer-associated antigens

Lymphocytes from lymph nodes draining the tumor region in patients with colorectal cancer were fused with two different human B-lymphoblastoid cell lines, LICR-LON-HMy-2 (HMy-2) and WI-L2-729-HF2 (729-HF2), to generate hybridomas synthesizing antibodies reacting with tumor-associated antigens. In this way 220 hybridomas were obtained which produce antibody reacting with colon cancer cells. All established clones produced IgM. Four human monoclonal antibodies have been further analyzed. The cell lines producing these antibodies are all hybrids based on DNA analysis. Three of the antibodies (G4146, B9165 and D4213) showed binding to differentiation antigens by immunocytochemical analysis on different cancer cell lines and normal human leucocytes and by immunohistochemical analysis on sections of frozen malignant and normal tissues, while the fourth (F11348) showed a reaction with all cells and tissues tested. Western blots of tumor extracts showed binding of G4146 to two components from colon cancer cells with Mr of 59 K and 61 K, while B9165 bound to a 43 K component and F11348 to several components with Mr from 30 to 200K. D4213 showed no binding in this analysis. The results obtained demonstrate the successful application of hybridoma technology to produce human monoclonals with reactivity to differentiation antigens.     

Schistosoma mansoni: Analysis of monoclonal antibodies reactive with gut-associated antigens

The analysis of a series of monoclonal antibodies (mAbs) developed in our laboratory against gutassociated antigens ofSchistosoma mansoni is described. It was found that mAbs that recognized epitopes of antigens in the gut and on the eggshell were mainly of the IgM isotype; these epitopes are likely to be carbohydrate in composition. Of a number of mAbs that were reactive with antigens important to the human humoral immune response, 75% appeared to be reactive with the circulating cathodic antigen.     

Studies of murine malaria antigens using monoclonal antibodies

A panel of ten monoclonal antibodies made againstPlasmodium chabaudi andPlasmodium yoelii infected mouse erythrocytes were used for characterization of antigens present in murine malaria. Screening of the antibodies in ELISA with different fractions of infected erythrocytes revealed both species-specific and fraction-specific monoclonal antibodies (MAbs), but also MAbs cross-reacting between the species. Two MAbs bound normal erythrocyte components. Subcellular localization of the target antigens was studied by immunofluorescence and their molecular identity by immunoblotting after SDS-PAGE. Of the MAbs toP. yoelii, one reacted with a cytoplasmic granule component of 137 k and two others reacted with vacuole-associated antigens of 26 k and 25/70/73 k, respectively. The latter antibodies cross-reacted withP. chabaudi antigens. Of the MAbs toP. chabaudi, all were species specific, one reacting with parasite surface antigens of 79 and 250 k and two with a vacuole-associated antigen of 70 k.     

Human monoclonal antibodies reactive with cell surface antigens on human leukemia cell lines: many antibodies are (auto)antibodies

Primary Epstein Barr Virus (EBV) transformants from peripheral blood mononuclear cells (PBM) established in macrocultures were screened for the secretion of antibodies reactive with cell surface antigens on one or another of two indicator human leukemic cell lines and fused with the HMMA2.11TG/O human fusion partner. Human monoclonal antibodies (HuMAbs) were readily obtained. Relative oligoclonality of the primary EBV macrocultures was documented by the number of antibody secreting hybridomas (1-100%). The method permitted preselection for fusion of transformants producing antibodies of certain specificities and/or class. Fourteen HuMAbs, primarily of the IgM class, have been obtained. Those IgM HuMAbs obtained from patients with active diseases, e.g. Acute Lymphoblastic Leukemia (3 HuMAbs), and HIV infection (4 HuMAbs), were found to have a relatively broad spectrum of reactivity with cell lines of various hematopoietic lineages and normal cells, although several show selective reactivity with T cell lineage tumors or a selected population of cells. HuMAbs from normal donors of both the IgG and IgM class were obtained. The IgM HuMAbs from one volunteer reacted primarily with autologous and allogeneic macrophages (autologous PBM from the other patients were not available) as well as a diverse number of hematopoietic cell lines. From others, the IgG HuMAbs demonstrated a more restricted spectrum of reactivity, while the IgM HuMAbs reacted with both autologous and allogeneic normal cells. Thus, the B cell repertoire contains cells capable of secreting cell surface reactive antibodies and many of these antibodies express characteristics of autoantibodies. Those that did not react with autologous or allogeneic PBM may react with other autoantigens which have been expressed on the malignant cells used as screening targets or may represent true antitumor antibodies.     

Serological and immunochemical characterization of monoclonal antibodies to Toxoplasma gondii.

The fusion of mouse NS-1 myeloma cells with spleen cells from mice chronically infected with Toxoplasma gondii resulted in eight clones of hybridomas producing monospecific antibodies against membrane or cytoplasmic antigens of Toxoplasma tachyzoites. One of the antibodies to a cytoplasmic determinant was an IgM; the others directed to membrane or cytoplasmic antigens belonged to the IgG2 or IgG3 isotypes. Antibodies of clones 1E11 (IgG3), 2G11, and 3E6 (IgG2) directed to membrane antigens, bound complement and were reactive in the complement-dependent cytotoxicity assay of Sabin-Feldman. These IgG2 antibodies were strongly agglutinating to parasites, whereas the IgG3 was relatively weak. Another IgG2 antibody (5B6), possibly recognizing a shared antigen of membrane and cytoplasm, exhibited a low titre in the cytotoxicity assay as well as in the agglutination assay. Two other antibodies to membrane antigens (2B7 and 2F8) as well as an antibody to a cytoplasmic antigen (3G3) did not bind complement and did not cause agglutination. The pattern of parasite staining produced by monoclonal antibodies to membrane antigens in an IFA test was different from that of polyvalent antisera. A strictly localized or 'beaded' staining was observed, as well as a smooth, rim fluorescence. Toxoplasma tachyzoites were surface radio-iodinated and the solubilized membrane proteins were immunoprecipitated with monoclonal antibodies and analysed by two-dimensional polyacrylamide gel electrophoresis. Two independently arising monoclonal antibodies to membrane antigens (2G11 and 3E6) consistently precipitated both the solubilized 35,000 and 14,000 mol. wt proteins, while 1E11 precipitated the 27,000 mol. wt protein.     

Three monoclonal antibodies defining distinct differentiation antigens associated with different high molecular weight polypeptides on the surface of human embryonal carcinoma cells

Two monoclonal antibodies (TRA-1-60 and TRA-1-81) recognizing distinct cell surface antigens on human embryonal carcinoma (EC) cells were produced and characterized. These antibodies reacted strongly with undifferentiated human EC cells in indirect radioimmunoassays (RIA) and immunofluorescence (IF) assays, but only weakly or not at all with cells derived from pluripotent EC cells differentiating in vitro or in xenograft tumors, nor with other germ cell tumor cell lines that did not also express the typical features of human EC cells. They did not react with murine teratocarcinoma cell lines. A survey of other human tumor cell lines and normal human tissues disclosed that molecules recognized by these antibodies are not confined to human EC cells but that cross-reacting epitopes appear on several neoplastic and normal tissues, although in a different anatomical pattern for each antibody. Both antibodies immunoprecipitated a major polypeptide (apparent molecular weight approximately 240,000) and a minor polypeptide (apparent molecular weight approximately 415,000) from lysates of 125I surface-labeled human EC cells, in this respect resembling another monoclonal antibody, 8-7D, previously described by Blaineau et al. (1,2) However, sequential immunoprecipitation revealed that each of the three antibodies reacted with different molecules of slightly different molecular weights. The epitopes defined by the present antibodies differ from those recognized by the other human EC cell-specific monoclonal antibodies that have been described and provide new markers for studying the differentiation of pluripotent human EC cells.     

Human monoclonal IgM antibodies with apoptotic activity isolated from cancer patients

Monoclonal antibodies are accepted as ideal adjuvant therapeutic reagents for all kinds of diseases. Polyvalent (cross-linking) and low-mutated IgM antibodies (less immunogenic) are believed to be the most effective weapons against cancer. The best sources for these types of antibodies are the cancer patients themselves. Using conventional hybridoma technology, not only are fully human monoclonal IgM antibodies isolated, but also new tumor-related targets can be identified using the same experimental approach. The resulting antibodies can be used directly for therapeutic purposes without further modulation and manipulation. This report describes five newly established human monoclonal IgM antibodies; antibody LM-1 that was isolated from a patient with lung cancer, antibodies PM-1 und PM-2 that were isolated from a patient with pancreatic cancer, and antibodies CM-1 and CM-2 which were isolated from a patient with colon carcinoma. The mainly germ-line encoded antibodies are specific for malignant tissues and show only restricted reactivity with healthy cells. When tested for in vitro functional activity, all five antibodies inhibit tumor cell proliferation of carcinoma cells by inducing apoptosis.     

Differential expression of carcinoembryonic antigens and non cross-reacting antigens in the human thymus. Analysis on frozen sections and cultured epithelial cells using monoclonal antibodies

The organization of epithelial cells in distinct areas of the thymus appears important for understanding the pathways of T-cell differentiation. The presence of carcinoembryonic antigen (CEA) was investigated on sections of human thymus and in cultures of thymic epithelial cells through use of monoclonal antibodies (Mab) discriminating CEA and 2 non cross-reacting antigens (NCA 55 and NCA 95). All together, 5 monoclonal antibodies were used. The Mab 35 and 73 recognized exclusively a specific CEA antigenic determinant, whereas Mab 47, 192 and 202 were also reactive with CEA cross-reacting antigens. By immunofluorescence or the immunoperoxidase technique, staining of CEA was restricted essentially to Hassall's corpuscles and a few adjacent epithelial cells on thymic sections; this pattern was similar to distribution of Ca 19-9 antigen. Additionally, the 47, 192 and 202 antibodies were reactive with a keratin-negative subset mainly located in the cortex. Furthermore, a subset of keratin-positive cells bearing CEA molecules was observed in thymic epithelial cell cultures. Positive cells comprised less than 3% with Mab 35 and 73, and reached up to 12% with Mab 47, 192 and 202. By flow cytometry analysis, staining intensity varied with the epitope; it was much weaker with Mab 35, 47 and 73 than with Mab 192 and 202. The CEA content in culture supernatants was inversely correlated to the number of CEA positive cells. Thus, CEA could be considered as a marker of a late maturation stage of medullary epithelial cells culminating in Hassall's corpuscles and could contribute to delineating the heterogeneity of thymic epithelial cells.     

Monoclonal antibodies defining a minor and a private idiotope on human rheumatoid factors

Two mouse monoclonal antibodies (mAb A216-5 and L 49-3) with antiidiotypic activity against two human monoclonal IgM rheumatoid factors (IgM RFs) were defined. Each of these monoclonal antibodies (two mouse IgG 1 K) reacted with an idiotope located on the heavy chain of the immunizing monoclonal IgM RF and was able to inhibit RF fixation to the antigen. These monoclonal antibodies did not react with other monoclonal IgM RFs from patients with macroglobulinemias or cryoglobulinemias and, therefore, did not recognize the known cross-reactive idiotopes of human monoclonal RFs. The presence of both 216-5 and 49-3 idiotopes on polyclonal IgM RFs from unrelated patients was undetectable by the inhibition assays. However, using a four-stage solid-phase radioimmunoassay, the 216-5 idiotope (minor), but not the 49-3 idiotope (private), was frequently present at a low concentration on polyclonal IgM RFs from patients suffering from rheumatoid arthritis, primary Sjögren syndromes, various infectious diseases, systemic vasculitis, and sarcoidosis and during aging. Interestingly, the 216-5 idiotope was undetectable among polyclonal IgM RFs of 12 normal adults. The main conclusions of these data are the following. (1) The definition of minor and private idiotopes of human RFs requires the use of assays able to detect low amounts of antibodies among polyclonal Ig. (2) The anti-IgG B cells which are sometimes clonally expanded during Waldenström diseases and cryoglobulinemias can also be activated during nonneoplastic diseases, among the other RF-secreting B cells. (3) Thein vivo expression of the IgM RF repertoire is different among normal adults and during various known IgM RF-inducing conditions.     

Diagnosis of human lymphoma with monoclonal antileukocyte antibodies

Two monoclonal antibodies have been produced that react with antigens present on human white cells. These reagents differ from other monoclonal antibodies of similar specificity in that the antigens they recognize are resistant to conventional tissue-fixation and embedding procedures. These reagents can therefore be used in immunocytochemical staining of paraffin-embedded tissue sections. We assessed the practical usefulness of this technique in the histopathological diagnosis of human lymphoid neoplasms by staining a wide range of routine surgical biopsy specimens of normal and neoplastic tissue (gathered from five institutions), using an indirect immunoperoxidase technique. In all 40 cases of non-Hodgkin's lymphoma, positive labeling of neoplastic cells was obtained with one or both antibodies. In contrast, no staining of neoplastic cells was observed in 60 samples of nonlymphoid neoplasms. We conclude that many of the difficulties encountered by histopathologists in distinguishing between lymphoid and nonlymphoid neoplasms may be overcome by immunohistologic labeling with monoclonal antibodies such as the ones we have studied.     

Reactivity of monoclonal antibodies against human leucocyte antigens with lymphocytes of non-human primate origin

The phylogenetic distribution of antigens present on human lymphocytes was investigated by incubating human or simian cells with murine anti-human monoclonal antibodies and then determining the level of reactivity with a radiolabelled anti-murine IgG reagent. The monoclonal antibodies used were specific for a T-cell antigen, lymphoid and lymphoid:myeloid antigens, Ia antigens, and beta 2 microglobulin. The cells examined included B- and T-lymphoblastoid cell lines and fresh peripheral blood lymphocytes separated by sheep erythrocyte rosetting into T-cell and non T-cell fractions. Results of these studies showed that the antibodies gave complete cross-reactivity with gorilla and chimpanzee cells while B-cell lines of orangutan origin had lost lymphoid and beta 2 microglobulin markers. Gibbon cells and cells of Old World and New World monkeys reacted strongly only with monoclonal antibodies against Ia antigenic determinants. These Ia antigens were found on the non T-cell fraction of fresh peripheral lymphocytes, on B-cell lines and on some virus induced T-cell tumour lines. Immunoprecipitation analysis using the anti-Ia antibodies showed a degree of molecular diversity on owl monkey and marmoset cells compared to the Ia antigens associated with human cells.     

Distinct epitopes on Ik gene products identified by monoclonal antibodies

Analysis of reactivity of monoclonal anti-Ia^k alloantibodies, obtained by fusion between NS 1 myeloma and spleen cells from alloimmune A.TH mice, permitted the identification of 9 distinct determinants of the I^k gene products. Competitive binding experiments indicated that 2 private epitopes (defined by H 8–109.13 and H 8–138.4 antibodies) of the I-A^k product could be separated, thereby apparently splitting the Ia.2 specificity. A public determinant of the I-A^k molecule (identified by H 8–15.9 antibody) was found expressed not only on the 1-A products of the H-2^b, H-2^d, H-2^ja, H-2^P and H-2^q murine haplotypes, but also on human HLA-DR antigens. Four determinants of the I-E/C^k antigen (defined by H 7–8.26, H 10–81.10, H 10–93.2 and H 8–86.2 antibodies) had a strain distribution analogous to the Ia.7 specificity. However, competitive binding experiments, and the cross-reactivity pattern with la-like antigens from other species (e.g. human HLA-DR antigens) indicated that these antibodies detected distinct determinants on the I-E/C^k molecule, thereby subdividing the broad Ia.7 specificity. Two other determinants (defined by H9–14.8 and H9–15.4 antibodies) had a strain distribution that did not permit a precise assignment to a given Ia antigen, even though preliminary data suggested that they could detect separate determinants on the I-E/C^k product. All these monoclonal antibodies identified membrane antigens with the expected Ia tissue distribution pattern, and most of them could precipitate a molecule containing two chains of 28 kD and 35 kD, from mouse spleen cell lysates.     

Use of monoclonal antibodies to identify shared idiotypes on anticardiolipin and anti-DNA antibodies in human sera.

Using hybridoma technology we produced monoclonal antibodies (MoAb) to idiotypic determinants on human anti-cardiolipin antibodies purified from a patient with SLE. Hybridomas were screened by inhibition of cardiolipin binding activity of sera from patients with SLE. Seven hybridomas were selected, two of which were studied extensively. Sera from a number of patients with SLE were found to share idiotypic determinants. This cross-reacting idiotype was not detectable on anti-cardiolipin antibodies in syphilis sera. The cross-reacting idiotype was present in sera with anti-ssDNA antibodies even though some of these sera had no anti-cardiolipin antibodies. We propose that these MoAb may recognize a regulatory idiotype.     

Species cross-reactive Ia determinants: detection with a monoclonal antibody to human Ia of a murine Ia. 7 epitope

A.TH anti-A.TL alloserum has previously been shown to react with monomorphic determinants of human Ia molecules. In an attempt to produce monoclonal antibodies detecting human-mouse cross-reacting epitopes, A.TH mice (Is) which lack I-E antigens were immunized with human Ia glycoproteins. Five hybridoma lines were established which recognize monomorphic human Ia determinants. Only one of the lines, 21w4, is reactive with murine cells and also with pig and sheep cells but not with rat cells. Binding studies to murine target cells show a strain distribution analogous to that of an Ia. 7-like specificity. The binding of 21w4 antibody to human and murine cells was compared to that of two other monoclonal antibodies directed to the murine Ia. 7-like specificity. Although all three antibodies had a similar pattern of reactivity with cells of different mouse strains, only 21w4 bound to the human cells tested. Competitive inhibition studies on murine cells have revealed that the epitopes were spatially related but not identical. Our results support the view that Ia. 7, the putative species cross-reacting specificity, might be composed of clusters of epitopes with variable degrees of conservation throughout evolution.     

J chain deficiency in human IgM monoclonal antibodies produced by Epstein-Barr virus-transformed B lymphocytes

Six human IgM monoclonal antibodies against Pseudomonas aeruginosa were purified and characterized. On agarose-acrylamide sodium dodecyl sulfate (SDS) gels run under nonreducing conditions, IgM monoclonal antibodies showed variable amounts of a slower migrating form of IgM in addition to the one co-migrating with plasma IgM. Protein blotting with anti-J chain antibody showed that the slower migrating form did not contain J chain. Analysis of one of the monoclonal antibodies by sucrose gradient centrifugation showed that the J chain-deficient form sedimented faster than the complete IgM. It is known that IgM preparations lacking J chain sediment faster by sucrose gradient centrifugation analysis and tend to form hexamers. The slower migrating form of IgM we observed on SDS gels under nonreducing conditions could be hexameric IgM. Further evaluation of this monoclonal antibody demonstrated that both forms of IgM had the same antigen-binding activity. Glycosylation of the light chain was demonstrated in two of the monoclonal antibodies.