Cytotoxicity of monoclonal antibodies against individually immunophenotyped human leukemic cells

Summary Peripheral blood and bone marrow mononuclear cells from 12 patients with acute myeloid leukemia (AML), 2 patients with acute lymphatic leukemia, and 1 patient with chronic myeloid leukemia in blastic crisis were taken at diagnosis or in relapse. Cells were immunophenotyped with a panel of monoclonal antibodies (Moab) (OKIa, Leu M1, Leu M2, Leu M3, Leu M4, B1, Okt 11, J5) and the same antibodies were used in an in vitro cytotoxicity test. Of the 14 patients, 10 had antibody-binding cells, and the percentage of lysed cells was almost equal to that of blasts. The other 4 patients had few binding cells and little lysis. Acute leukemia with and without preceding myelodysplastic features did not differ in immunophenotype. Mean spontaneous release of ⁵¹Cr was 12.7% and complement alone caused an additional average release of 11.8%. Four single antibodies together with complement showed a mean ⁵¹Cr release of 0.7–32.4% above that found with complement alone. Combinations of Moabs resulted in ⁵¹Cr release at least 10% above the single most efficient Moab in 8 out of 12 patients. Not all blast cells showed antibody binding, nor were all antibody-binding cells susceptible to cytotoxicity.Normal bone marrow growth in vitro seemed to be stimulated by factors in complement and in the Moab. When this stimulation was compensated for by adding fetal calf serum, cytotoxicity tests prior to CFUc assays resulted in a mean decrease of 46% of colonies and 25% of clusters in normal bone marrow. CFUc are thus sensitive to the cytotoxicity, although CFU may also be resistant.Supported by Sw. Cancer Res. Found. Grant no. 699-B83-19XC Preliminary report given to Int. Soc. Hematol., Eur. Afr. Div. 5, 1983 Visiting scientist from Hacettepe University Ankara, TurkeyVisiting scientist from Hacettepe University Ankara, Turkey     

Differentiating between isolates of Vibrio vulnificus with monoclonal antibodies

Monoclonal antibodies (MAbs) against Vibrio vulnificus (isolate I, VVC and isolate II, VVB) were raised using heat-killed and heat-killed plus SDS-mercaptoethanol treated forms of VVC and VVB for immunizing Swiss mice. Twenty three hybridomas producing MAbs against V. vulnificus were selected and divided into five groups according to their specificities to different V. vulnificus isolates and apparent protein antigens which ranged from approximately 3-50 kDa. Four groups were specific to V. vulnificus without cross reactivity to either other Vibrio spp. or other bacterial species. In dot blot based assays, one group of MAbs were specific to VVC, with a sensitivity of approximately 1.6 x 10(7) CFU ml(-1) (approximately 1.6 x 10(4) cells spot(-1)), and bound to proteins of approximately 50 and approximately 39 kDa. Other MAbs, binding to proteins ranging from approximately 3-14 and approximately 40 kDa, detected VVB (but not VVC) with high sensitivity at approximately 1.6 x 10(5) and 4 x 10(6) CFU ml(-1) (approximately 1.6 x 10(2) and 4 x 10(3) cells spot(-1)), respectively. In addition, certain MAbs were able to recognize V. vulnificus in tissues by means of immunohistochemistry. The remaining groups demonstrated cross reactivity to Vibrio fluvialis. MAbs from this study can, therefore, detect the difference between some isolates of V. vulnificus and in addition to pathogen detection may, with further antibodies, form the basis of serovar typing isolates in the future.     

Differentiating between isolates of Vibrio vulnificus with monoclonal antibodies

Monoclonal antibodies (MAbs) against Vibrio vulnificus (isolate I, VVC and isolate II, VVB) were raised using heat-killed and heat-killed plus SDS–mercaptoethanol treated forms of VVC and VVB for immunizing Swiss mice. Twenty three hybridomas producing MAbs against V. vulnificus were selected and divided into five groups according to their specificities to different V. vulnificus isolates and apparent protein antigens which ranged from ∼ 3–50 kDa. Four groups were specific to V. vulnificus without cross reactivity to either other Vibrio spp. or other bacterial species. In dot blot based assays, one group of MAbs were specific to VVC, with a sensitivity of ∼ 1.6 × 10⁷ CFU ml^− 1 (∼ 1.6 × 10⁴ cells spot^− 1), and bound to proteins of ∼ 50 and ∼ 39 kDa. Other MAbs, binding to proteins ranging from ∼ 3–14 and ∼ 40 kDa, detected VVB (but not VVC) with high sensitivity at ∼ 1.6 × 10⁵ and 4 × 10⁶ CFU ml^− 1 (∼ 1.6 × 10² and 4 × 10³ cells spot^− 1), respectively. In addition, certain MAbs were able to recognize V. vulnificus in tissues by means of immunohistochemistry. The remaining groups demonstrated cross reactivity to Vibrio fluvialis. MAbs from this study can, therefore, detect the difference between some isolates of V. vulnificus and in addition to pathogen detection may, with further antibodies, form the basis of serovar typing isolates in the future.     

Uses and limitations of monoclonal antibodies to Giardia lamblia-specific 66-kDa copro-antigen in copro-immunodiagnosis of giardiasis

Abstract Antigen-capture enzyme-linked immunosorbent assay for the detection of Giardia lamblia -specific antigen in stool eluates from clinical subjects employing monoclonal antibody directed at 66-kDa G. lamblia copro-antigen has been evaluated. The G. lamblia copro-antigen was detected in 67% (31 of the 46 cases) of stool eluates from clinical cases, while none of the stool eluates from subjects with other intestinal parasites or from apparently healthy individuals, had detectable levels of G. lamblia copro-antigen. Monoclonal antibodies secreted by clones B4C5 and D3F4 recognised the periodate-sensitive and -insensitive epitopes of 66-kDa G. lamblia specific copro-antigen, respectively. Eight (73%) of the 11 symptomatic cases of giardiasis had trypsin-/periodate-sensitive epitopes of 66-kDa copro-antigen while 9 (92%) of 11 of the symptomatic cases and asymptomatic G. lamblia cyst carriers had trypsin-sensitive periodate-insensitive G. lamblia specific copro-antigen. The data tend to suggest that detection of periodate-insensitive epitopes of G. lamblia copro-antigen would indicate the presence of the parasite while the detection of periodate sensitive epitopes of G. lamblia copro-antigen would suggest symptomatic active giardial infection.     

Human monoclonal anti-La antibodies. The La protein resides on a subset of Ro particles

We have addressed the problem of anti-La autoimmune responses by defining the specific binding sites of human mAb to the La protein. Two human anti-La mAb were developed; one an IgM (kappa) (designated 8G3) and the second an IgG1 (kappa) (9A5) isotype. The mAb 8G3 immunoprecipitated the La RNA and La protein from crude human cell lysates; bound the 50-kDa La protein and a 28-kDa digestion fragment in immunoblots, and recognized a small defined internal segment from the cloned La protein. In contrast, the IgG isotype (9A5) failed to precipitate native La from cell lysates but bound the same segment of digested La protein and the same polypeptide of 131 amino acids in length from the cloned La protein. Immunoprecipitation experiments performed with these mAb demonstrated that the La protein is a component of a subset of Ro particles. The data suggest that the La protein is not present on the hY RNA in the absence of the Ro polypeptide. These observations may define functional subsets or maturation states of hY RNA based on their association with Ro or Ro and La polypeptides.     

Mapping of monoclonal antibodies specific to P64k: a common antigen of several isolates of Neisseria meningitidis

P64k is a minor outer membrane protein from Neisseria meningitidis. This protein has been produced at high levels in Escherichia coli. We generated a group of monoclonal antibodies (mAbs) against recombinant P64k, which recognise four non-overlapping epitopes, as shown using competition assays with biotinylated mAbs. The P64k sequences involved in mAbs binding were mapped with synthetic overlapping peptides derived from the P64k protein, and located in the previously determined three-dimensional structure of the protein. These antibodies were also characterised by whole-cell ELISA and bactericidal tests against N. meningitidis. Only two of the recognised epitopes were exposed on the bacterial surface, and none of the mAbs showed bactericidal activity. The relationship between these results and the structural data on the epitopes bound by the mAbs is discussed.     

A monoclonal antibody to a cross-reactive idiotype on cationic human anti-DNA antibodies expressing lambda light chains: a new reagent to identify a potentially differential pathogenic subset

Anti-double-stranded DNA antibodies are commonly found in the serum of patients with systemic lupus erythematosus (SLE). They are a heterogeneous group of antibodies thought to differ in pathogenicity. The degree of heterogeneity and the structural correlates of pathogenicity, however, remain poorly defined. To address these questions we have been generating anti-idiotypic antibodies to the anti-DNA antibodies found in the serum of SLE patients. In this paper we report the generation and characterization of a new murine monoclonal anti-idiotype, 8.12, that recognizes a subset of anti-DNA antibodies that is present in serum of approximately 50% of patients with SLE. The 8.12 anti-idiotype recognizes uniquely cationic anti-DNA antibodies, all of which express lambda light chains. In murine models of SLE, it has been suggested that cationic anti-DNA antibodies are preferentially deposited in the kidney. It may be, therefore, that 8.12 recognizes a subset of anti-DNA antibodies of particular pathogenic significance.     

Characterization of monoclonal antibodies to bromodeoxyuridine

The characteristics of three mouse monoclonal antibodies to halogenated uridine derivatives are presented. Two, IU-1 and IU-2, are produced by hybridomas derived in our laboratory, and the third is the B-44 hybridoma described by Gratzner (7) and obtained commercially from Becton-Dickinson Monoclonal Center. Hybridomas IU-1 and IU-2 were derived from the fusion of spleen cells from a Biozzi High Responder mouse immunized with iododeoxyuridine (IdUrd) conjugated to bovine serum albumin and SP2/0 mouse myeloma cells. This paper presents methods and results for enzyme-linked immunosorbent assays (ELISA) against whole cells labeled with bromodeoxyuridine (BrdUrd), ELISA against BrdUrd-labeled DNA, and a competition ELISA for free BrdUrd. All three antibodies show similar binding affinities and specificities. The IU antibodies react with BrdUrd and IdUrd when the nucleosides are either free in solution or incorporated into single-stranded DNA (ss-DNA). The antibodies do not recognize either halogenated base in double-stranded DNA (ds-DNA), nor do they react with uracil or bromocytidine. Weak binding to thymidine, 5-fluorodeoxyuridine, and unsubstituted ss-DNA occurs.     

Characterization of monoclonal antidigoxin antibodies immobilized to a solid support

A high-affinity monoclonal antidigoxin antibody, produced by somatic cell fusion, was amplified by the formation of ascites. Purification from ascites was accomplished by affinity chromatography by passing the ascites over a digitoxin-amine-agarose column. Affinity-purified antidigoxin antibody was coupled to a pellicular microbead at concentrations of 10, 25, 50, and 100 mg/g bead. The immobilized antibody was characterized for binding affinity, for specificity to other cardiac glycosides, and for binding capacity. There were no changes in the binding affinity observed for the immobilized antibody when compared to that of the antibody grown in culture media. Binding capacities for the immobilized antibody were decreased from calculated theoretical values. Saturating the microbead with increasing concentrations of antibody lowered the binding efficiency of the antibody from 32 to 22% of theoretical values. Attempts to improve the binding capacity by immobilizing antibodies to the microbead at the immunoglobulin carbohydrate by periodate oxidation were unsuccessful. These data demonstrate that antidrug antibodies immobilized on solid supports remain functional and may have the capability of removing drug from biological fluids passed over the support.     

Characterization of antibodies to idiotypic determinants of monoclonal anti-sperm antibodies

Rabbit polyclonal antibodies to the idiotype of murine monoclonal anti-sperm antibodies were developed and characterized. M29.6 and M42.15 are monoclonal antibodies (mAbs) that inhibit fertilization in vivo and in vitro. Sera from rabbits inoculated with purified mAbs (Ab1) were absorbed with normal mouse and isotype-specific immunoglobulin (Ig); the anti-idiotype Ig fraction (Ab2) was isolated by protein A-chromatography or by chromatography on the corresponding idiotype column. Binding specificity of Ab2 was confirmed by measuring the reactivity of Ab2 with homologous and heterologous mAbs. Ab2 competitively inhibited 125I-labeled Ab1 binding to mouse sperm, suggesting that the Ab2 preparation possessed subpopulations directed against idiotopes similar or adjacent to the antigen-binding site of the mAb. Anti-idiotype antibodies reactive with the antigen-combining site of the anti-sperm mAb may contain subpopulations that mimic the mouse sperm epitope recognized by Ab1. Immunization with Ab2 induced anti-(anti-idiotype) antibodies (Ab3), which competitively inhibited binding of 125I-labeled Ab1 to immobilized Ab2. These results are consistent with the hypothesis that immunization of mice with antibodies to the idiotype of sperm-specific mAbs can induce antibodies that share structural similarities with the anti-sperm mAb used for their induction. Immunization with anti-idiotype antibodies that mimic sperm antigen structure represents a possible strategy for induction of immunity to sperm.     

A morphometric comparison of five axenic Giardia isolates

The length and width of trophozoites from axenic cultures of 5 Giardia isolates were measured both live and after fixation and Giemsa staining. These isolates, as named on the basis of host source, are classified as G. lamblia (3 isolates), G. felis (1 isolate), and G. caviae (1 isolate). The size of live, unstained trophozoites from the 5 isolates, measured without regard to the presence or absence of median bodies, showed only occasional significant differences in length. Statistically significant differences in length and/or width were observed for all comparisons when stained preparations of the isolates were compared. These size differences occurred between isolates assigned to different species as well as among the 3 G. lamblia isolates. These data and previously reported isozyme studies of these isolates most appropriately led to a re-examination of the presently utilized criteria for Giardia speciation.     

Characterization of syngeneic rat monoclonal antibodies to the HSN tumor using syngeneic monoclonal anti-idiotopic antibodies

Twelve syngeneic anti-idiotopic mAb (anti-idiotypic/idiotopic antibodies Ab2)) were prepared from CBH/Cbi rats immunized with one of three monoclonal anti-HSN antibodies (Ab1) (11/160, ALN/11/53, or ALN/16/53) specific for the HSN tumor. The sera of the rats used for hybridoma production and all of the monoclonal Ab2 specifically inhibited the binding to HSN of the immunizing Ab1 only. It is concluded that, in this completely syngeneic system, only the private idiotopes associated with the antibody-combining site were immunogenic. Analyses using Western blotting showed that the Ab2 bound to intact Ab1 and to isolated H chains where the intra-strand disulfide bonds remained intact. The Ab2 did not bind to L chains or to fully reduced H chains of the Ab1. It is concluded that the idiotopes expressed on the H chain were conformational. When a panel of 13 monoclonal Ab1 (including the three used for immunization) were tested for reactivity with the Ab2, three reacted specifically with their respective Ab2 and 8 gave no binding suggesting that each Ab1 had a distinct idiotypic specificity despite the fact that all the Ab1 competed with each other for binding to Ag. However, the two remaining Ab1 (ALN/9/94 and ALN/12/17) generated from different tumor-bearing rats, were found to possess the same idiotypic specificity as 11/160. A detailed analysis using seven Ab2 raised against 11/160 showed that while the idiotype of ALN/9/94 and 11/160 were very similar, that of ALN/12/17 showed some clear differences. These three Ab1 have been shown previously to bind a sequential epitope on the HSN Ag in Western blots and it is postulated that the common idiotype of these Ab1 reflects their recognition of a sequential epitope. This may also account for the relatively frequent occurrence in tumor bearer sera of antibodies with this Id.     

Autoimmune syndrome after induction of neonatal tolerance to alloantigens: effects of in vivo treatment with anti-T cell subset monoclonal antibodies

BALB/c (H-2d) mice rendered tolerant to h-2b alloantigens by neonatal injection of semiallogeneic (C57BL/6 X BALB/c)F1 spleen cells develop autoimmune features due to an abnormal activation of persisting F1 donor B cells. The role of T cells in this autoimmune syndrome was studied by in vivo treatment of tolerant mice with anti-L3T4(GK-1.5) or anti-Ly-2 (H-35-17.2) monoclonal antibodies. The treatment of tolerant mice from day 2 to day 21 of life with anti-L3T4 MAb completely prevented the occurrence of circulating immune complexes of anti-ssDNA anti-Sm and anti-hapten (FITC) IgG antibodies as well as the glomerular deposition of Ig that were usually seen in untreated tolerant mice. This effect persisted for at least 6 wk after stopping this treatment. When the injections of anti-L3T4 MAb were delayed until day 15 of life, a very significant decrease of the autoimmune manifestations was still observed. Treatment of tolerant mice with anti-Ly-2 MAb during the same period had no effects on the autoimmune disease as compared with untreated tolerant mice. No effects on the maintenance of tolerance vs H-2b alloantigens were observed after treatment with anti-L3T4 MAb, as followed by the decrease of CTL and CTL-p alloreactivity and by the persistence of F1 donor B cells, indicated by the presence of Ig bearing the Ighb donor allotype. These results suggest the existence of interactions between L3T4+ T cells and persisting autoreactive B cells from F1 donor origin in the development of the autoimmune syndrome after neonatal induction of transplantation tolerance.     

Reaction patterns of monoclonal antibodies to DNA

Starting with spleen cells from MRL/lpr, NZB/W, and graft-vs-host-diseased mice, we prepared a total of 57 hybridomas that produce antibodies to DNA. Using various approaches, we studied the avidity of these monoclonals in relation to their behavior in four anti-DNA assays. From the results obtained, we postulate that on the basis of anti-DNA avidity the anti-DNA ELISA, the polyethylene glycol assay, the indirect immunofluorescence test on Crithidia luciliae, and the Farr assay (in this order) detect a decreasing amount of anti-dsDNA, the Farr assay being strictly selective for high avidity anti-dsDNA. mAb selected by the anti-DNA ELISA generally were of a low avidity toward DNA. Using cardiolipin and dextran sulfate, a polyanion that bears a resemblance in charge to DNA, we studied the cross-reactivity of the monoclonals. A total of 6 of the 57 monoclonals were found to cross-react with cardiolipin, and 26 with dextran sulfate. We observed an inverse relationship between anti-DNA avidity and cross-reactivity: the lower the avidity of the antibody, the more cross-reactive it is. Based on these findings, we postulate that it is at least questionable whether low avidity, cross-reactive (monoclonal) anti-DNA is representative for the anti-DNA found in patients with SLE.     

Characterization of monoclonal antibodies to Treponema pallidum

Thirteen hybrid cell lines which produce mouse monoclonal antibodies to Treponema pallidum, the causative agent of syphilis, have been established. All of the monoclonal antibodies react with T. pallidum, Nichols strain, in ELISA and in immunofluorescence assays, but do not react with normal rabbit testicular tissue in the ELISA. Two of these antibodies were demonstrated to react with the nonpathogenic treponemes T. phagedenis, biotype Reiter, T. refringens (Noguchi strain), T. vincentii, and T. denticola (strains 11 and W), as well as with Borrelia recurrentis, Leptospira interrogans, serogroup Canicola, and the swine pathogen T. hyodysenteriae. The remaining 11 antibodies react with four recently isolated strains of T. pallidum, but with none of the related nonpathogens nor with Borrelia or Leptospira. Thus, our results to date indicate that these monoclonal antibodies may identify antigenic determinants that are specific either for T. pallidum alone or for those treponemes which are pathogenic for humans. The molecular specificities of six of the 13 antibodies were determined by Western blotting. We anticipate potential usefulness of these antibodies in the investigation of the antigenic structure of T. pallidum, the taxonomic study of the pathogenic and nonpathogenic treponemes, and in the diagnosis of syphilis.     

Cytotoxicity of a conjugate of ricin A-chain and monoclonal anti-allotypic antibody

A conjugate was developed between ricin A-chain and monoclonal antiallotypic antibody specific for L-chains of the rat Ig. Incubation of spleen cells with the conjugate (10(-7) M) resulted in a strong decrease of 1a-positive B-lymphocyte growth. At the same time spleen cells that lacked this antigen were not affected by immunotoxin (IT). Both the isolated antibodies and ricin A-chain did not inhibit cell growth in the same concentrations. IT injection to 10-day-old heterozygous rats (1a/1b) resulted in a partial suppression of Ig production with 1a-allotype. The conjugate was 4.5 times more effective than the isolated antibodies used to form it. The in vivo and in vitro differences in IT cytotoxicity were probably connected with insufficient efficacy of conjugate targeting in vivo.     

A comparison of isozymes of five axenic Giardia isolates

The relative mobilities of six enzymes from the trophozoites of five axenically-cultured isolates of Giardia from human, cat, and guinea pig hosts were compared by starch and polyacrylamide gel electrophoresis. The six enzymes compared were malate dehydrogenase (NAD+) (MDH) (EC 1.1.1.37), malate dehydrogenase (decarboxylating) (ME) (EC 1.1.1.40), hexokinase (EC 2.7.1.1), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), glucose-6-phosphate dehydrogenase (G6P) (EC 1.1.1.49), and alpha-glycerophosphate dehydrogenase (EC 1.1.1.8). The latter three enzymes have not been previously reported in Giardia. On the basis of zymogram patterns, the five Giardia isolates were divided into three zymodemes. Zymodeme I comprised human-1/England, human-1/Bethesda, and cat-1/Portland, Zymodeme II the guinea pig-1/Portland isolate, and Zymodeme III the human-1/Portland isolate. These zymodemes were further substantiated when several physical and kinetic properties of three of the enzymes, MDH, ME, and G6P, were examined. Our results, in which Giardia isolated from different mammalian hosts share multiple isoenzymes, question the validity of the practice of assigning Giardia species names on the basis of the animal host from which the protozoan was obtained.