Monoclonal antibodies as probes of tetanus toxin structure and function

Monoclonal antibodies specific for fragment B, fragment C, and light chain of tetanus toxin were prepared by fusion of P3X63Ag8 BALB/c myeloma cells with spleen cells from BALB/c mice immunized with tetanus toxoid or fragment B. Hybridoma colonies were assayed for antibody production by an enzyme-linked immunosorbent assay. Fourteen positive clones were identified, cloned by limiting dilution, and injected intraperitoneally into mice to obtain ascites fluids. Thirteen of the monoclonal antibodies were of the immunoglobulin G1 subclass and one was immunoglobulin G2. Two of the antibodies were directed against sites on fragment C, nine were directed against the light chain, and three were directed against the portion of fragment B which does not comprise the light chain of tetanus toxin. At least one antibody in each group exhibited significant toxin neutralization activity. However, only one of these neutralizing antibodies strongly inhibited the binding of 125I-tetanus toxin to ganglioside-coated plates. These data indicate that interference with receptor recognition is not the only means of neutralizing tetanus toxin. Monoclonal antitoxins as potential therapeutic and prophylactic reagents are discussed.     

Monoclonal antibodies against ricin: effects on toxin function

Monoclonal antibodies against ricin toxin were produced and some of their properties investigated. Antibodies 196 C12 and 197 C7 raised against A-chain reacted with a CnBr fragment probably comprised between amino acid 254 and 262. Antibodies 193 A9, 196 A3, and 191 B7 recognized a 6-7 kD CnBr peptide. A second set of antibodies was raised against whole inactivated ricin. Most of them bound in a solid phase radioimmunobinding assay only to ricin and few had a low activity against purified A-chain. Different effects were noted on toxin action in cultured leukemic cells. If cells were preincubated with ricin followed by antibodies, MAb 207 E5 and 216 B3 had a strong enhancing effect on toxin action. If antibodies and toxin were mixed and then added to sensitive cells, antibody 207 E5 gave a strong protection while 216 B3 maintained its enhancing activity. The effect of antibody 216 B3 was further investigated by quantitative cloning experiments which showed that toxin had a fivefold enhancement in its activity by a preincubation with this antibody. Binding of fluoresceinated ricin to leukemic target cells was inhibited by a preincubation with antibody 207 E5 while antibody 216 B3 had no effect.     

Monoclonal antibodies that inhibit ADP-ribosyltransferase but not NAD-glycohydrolase activity of pertussis toxin

Kenimer et al. (J. G. Kenimer, J. Kim, P. G. Probst, C. R. Manclark, D. G. Burstyn, and J. L. Lowell, Hybridoma 8:37-51, 1989) identified three classes of monoclonal antibodies, termed A, B, and C, that recognize the S1 subunit of pertussis toxin. This report presents data demonstrating that class A monoclonal antibodies (3CX4, 6D11C, and 3C4D), which block the ADP-ribosyltransferase activity and recognize the predominant neutralizing epitope on the S1 subunit of the toxin, do not inhibit the NAD-glycohydrolase activity of the toxin. In addition, alkylation of cysteine 41 of the S1 subunit, which may interact with NAD, inactivates the toxin but does not prevent binding by class A antibodies. Taken together, these results support the conclusion that proper alterations of amino acids that interact with NAD should allow for inactivation of the toxin without destruction of the predominant neutralizing epitope. The class A antibodies recognized control but not heat-treated pertussis toxin spotted onto nitrocellulose, indicating that class A antibodies do not recognize denatured S1 subunit. In contrast, a nonneutralizing class C antibody (X2X5) failed to bind to control toxin or S1 subunit in solution and recognized heat-treated pertussis toxin better than control toxin when spotted onto nitrocellulose. Thus, this type of analysis presents a heterogeneous mixture of fully or partially denatured and native S1 proteins and fails to distinguish between neutralizing and nonneutralizing antibodies.     

Monoclonal antibody against pertussis toxin: effect on toxin activity and pertussis infections.

Antibody-producing hybridomas of myeloma SP2/O and spleen cells of BALB/c mouse immunized with pertussis toxoid and pertussis toxin were selected by the binding ability of the monoclonal antibody to the subunit protein of the toxin. Two monoclonal antibodies, 1B7 and 3F10, specific for a subunit which has no binding activity to haptoglobin and sheep erythrocytes, named S1, and one antibody, 1H2, for a subunit related to the binding activity of the pertussis toxin molecule to haptoglobin or sheep erythrocytes, named S4, were examined for mouse protective activity against pertussis infection. Antibody 1B7 not only neutralized leukocytosis-promoting and islet-activating activities of the toxin but also protected mice against intracerebral and aerosol challenge with Bordetella pertussis. The antibody, furthermore, showed therapeutic effects on mice showing severe clinical signs with pertussis infection. The other two antibodies, 3F10 and 1H2, showed neither neutralizing nor protecting activity, nor significant synergistic effects on antibody 1B7.     

Monoclonal antibodies that define neutralizing epitopes of pertussis toxin: conformational dependence and epitope mapping.

The epitope specificities of 13 hybridomas secreting monoclonal antibodies (MAbs) specific for pertussis toxin (PT) is described. Hybridoma lines were derived by the fusion of spleen cells from mice immunized with native PT, Formalin-detoxified PT, or isolated PT subunits (S1 to S5) with the myeloma line X63-Ag8.653. Five MAbs showed a toxin-neutralizing ability, which was demonstrated by use of a Chinese hamster ovary cell assay system and by a NAD glycohydrolase assay. All five toxin-neutralizing MAbs demonstrated high specificities for and reactivities with native PT but were unable to bind to denatured PT. One MAb was able to neutralize the enzymatic activity of PT. The other four neutralizing MAbs inhibited the binding of PT or PT subunits to the surface of Chinese hamster ovary cells, as shown by an immunofluorescence assay. All neutralizing MAbs reacted with purified S2-S4 or S3-S4 dimers but not with S4 alone. Three MAbs which recognized a common epitope shared by S2 and S3 (which are about 70% homologous at the DNA level) and one MAb which recognized S4 were not neutralizing. Isolated S2-S4 and S3-S4 dimers bound to Chinese hamster ovary cells. These results indicate that the majority of critical epitopes which elicit neutralizing antibody are conformation dependent.     

Monoclonal antibody that neutralizes pertussis toxin activities

Pertussis or whooping cough is a disease with high mortality among infants and small children. The disease is caused by infection of the respiratory tract by a gram negative bacterium, Bordetella pertussis. The superficial colonized bacteria produce a myriad of toxins which enter the circulation causing various pathophysiologicalal changes in the host. Although antimicrobial therapy reduces the number of the coughed out bacteria and also the infectious time of the infected host, but it is not effective in amelioration of the clinical manifestations as the pertussis morbidity is due principally to the pertussis toxin (PT). Antibody based-therapy is frequently practiced in conjunction with other supportive measure to resuscitate the patient. Nevertheless, human derived antiserum against PT is of the limited supply and the ethical concern. Thus in this study a hybridoma clone, i.e. clone PT6-2G6, secreting monoclonal antibody (MAb) specific to the S1 subunit, the active enzyme of the PT that intracellularly ADP-ribosylates the host Gi-protein, was produced. The MAbPT6-2G6 inhibited the in vitro hemagglutination of chicken erythrocytes which is the activity of the B oligomer of PT; thus we hypothesize that the MAb bound to its epitope on the S1 subunit and stereologically hinders the binding sites of the B subunits. The MAb also inhibited ex vivo Chinese hamster ovarian cell clustering and neutralized the in vivo leucocytosis- promotion in mice which are usually mediated by intracellular S1 subunit. The large molecular nature of the intact MAb and its molecular hydrophilicity led us to speculate that the observed PT neutralizing activities of the MAb were due to interfering with the cellular entry of the S1 rather than the intracellular enzyme neutralizing activity per se. While further experiments are needed to pinpoint the MAb neutralizing activity and to identify the amino acid sequence and location of the MAbPT6-2G6 epitope, our findings indicate that this murine MAb, in its humanized-version, should have high therapeutic potential for pertussis.     

Monoclonal antibodies as structural probes of southern bean mosaic virus

The reactivity of six monoclonal antibodies with native southern bean mosaic virus particles, swollen virus, swollen virus contracted by divalent cations or by pH adjustment to 5.0, and virus coat protein at pH 7.5 and 5.0 were assessed by antigen inhibition enzyme-linked immunosorbent assays. Clones B4, B7, and B11 did not react with native virus but were inhibited by low levels of all nonnative virus antigens. B6 had a high reactivity only with the native virus. B5 and B10 reacted equally with native virus and swollen virus contracted by Ca2+ or by pH adjustment, but reacted only weakly with swollen virus or swollen virus contracted by Mg2+. B5 reacted with viral protein at pH 5.0 and weakly with coat protein at pH 7.5, whereas B10 did not react with protein at pH 7.5 and had only limited reactivity with viral protein at pH 5.0. The reactivity of B5 and B10 with swollen virus increased as the pH was gradually decreased between 7.3 and 6.5. Similarly, their reactivity with swollen-contracted virus decreased as the pH was increased within the same range. Reactivity versus pH plots of these two transition forms showed hysteresis with B10 but did not with B5.     

Recognition of pertussis toxin by antibodies to synthetic peptides

Eight synthetic peptides, selected from the amino acid sequence of pertussis toxin (PT) subunits S1, S2, S3 and S4, were assessed for their ability to induce protein-recognizing and neutralizing antibodies. Seven of these peptides, prepared as conjugates of either keyhole limpet haemocyanin or tetanus toxoid, induced significant levels of antibody, all of which reacted with SDS-denatured PT on Western blots. Six of the antibodies bound to PT-coated ELISA plates; this binding was inhibited by homologous peptide antigen. However, none of the antibodies, including those directed against the N-terminus of subunit S1, were able to attenuate in vivo or in vitro toxin-dependent activity. Further investigation revealed that only one antibody, specific for the C-terminus of S1 (peptide Slc, 237-255), could recognize the conformation of native PT in solution. The other five antipeptide antibodies which reacted with PT-coated ELISA plates did not recognize PT when captured onto ELISA plates via either a monoclonal antibody or fetuin, unless the conformation of the toxin had been relaxed by reduction with dithiothreitol. Conversely, the native PT-recognizing response of peptide Slc did not bind the conformationally relaxed PT molecule. From this study, it appears likely that a peptide capable of inducing PT-neutralizing antibody must closely resemble the conformation of the cognate sequence in the native protein.     

Monoclonal antibodies against tetanus toxin and toxoid

Monoclonal antibodies against tetanus toxin and its toxoid were produced by immunizing mice with toxoid or toxin. They were measured by an enzyme-linked immunosorbent assay (ELISA), by a toxin neutralization test in mice (in vivo prevention test), and by their ability to prevent binding of¹²⁵I-toxin to brain membranes or gangliosides (in vitro prevention test). Six monoclonal antibodies obtained by immunization with toxoid (anti-toxoid 1–6) were investigated in more detail. They belonged to IgG class 1. Three of them (anti-toxoid 1, 2 and 3) recognized both toxoid and toxin as well as fragment B and the light chain of toxin, but not fragment C. Two other antibodies (anti-toxoid 4 and 5) were directed against toxoid only. Neither of them prevented toxin action in vitro or in vivo. Anti-toxoid 6 recognized toxin, toxoid and fragment C, but not light chain, and prevented toxin action in vitro and in vivo. Immunization against toxin was initiated with a toxin-antitoxin complex and boosted with toxin. We studied six antibodies in more detail, all of IgG type 2. Their K_D against¹²⁵I-tetanus toxin varied from 10^–9 to 10^–10 M. Anti-toxin 2 recognized toxin, toxoid, light chain and fragment B, but not fragment C. The others reacted with toxin, toxoid and fragment C, but not with light chain or fragment B. All of them prevented toxin action in vitro and in vivo. As calculated from the maximal extinction achieved in the ELISA, tetanus toxin combined with a maximum of two different antibody molecules from our set. Gel filtration data indicate that tetanus toxin reacts with monoclonal antibodies one by one. Compared with polyclonal antiserum, monoclonal antibodies yield flatter slopes in both in vitro and in vivo prevention tests. Thus, they cannot substitute for the polyclonal antibodies in clinical situations, and cannot be calibrated in international units.This communication contains parts of the M.D. thesis of K.G. and C.V.     

Immunoglobulin A antibodies to pertussis toxin and filamentous hemagglutinin in saliva from patients with pertussis.

Immunoglobulin A (IgA) antibodies against pertussis toxin (PT) and filamentous hemagglutinin (FHA) in 181 saliva samples obtained during various stages of pertussis from 112 patients were determined. Saliva samples obtained within 5 days after the onset of symptoms did not have detectable IgA antibodies against either of the two antigens. Of the samples obtained between 6 and 50 days after the onset of symptoms, 72% had antibodies against FHA but only 40% had antibodies against PT. With few exceptions, saliva samples obtained more than 50 days after the onset of symptoms contained antibodies against both antigens. In the 59 patients from whom paired saliva samples were obtained at intervals of 2 to 5 weeks, a significant increase in the geometric mean FHA antibody titers but not PT antibody titers occurred. However, increases that were fourfold or greater were observed against FHA in only 19 patients and against PT in 14 patients. Thus, IgA antibodies against FHA and PT in saliva develop during pertussis, and the importance of secretory IgA antibodies for protection against infection and disease should be investigated. Determination of these antibodies in paired saliva samples is, however, of little value for the laboratory diagnosis of pertussis.     

Monoclonal antibodies to CD3 as immunosuppressants

Murine monoclonal antibodies specifically directed at the CD3 molecular complex are widely used in clinical transplantation. They are potent inhibitors of immune function and may be administered in association to conventional immunosuppressants. The main purpose of this review is to summarize our present knowledge on the CD3 molecule, to discuss the available data on the clinical use of anti-CD3 MAb (therapeutic effectiveness, mode of action and side effects) and the recent information derived from the use of anti-CD3 MAb in experimental models that are opening new perspective in the applications of anti-CD3 and will hopefully lead to the extension of its clinical use to settings different from transplantation.     

Interaction of monoclonal antibodies with pertussis toxin and its subunits

The pathogenicity of Shigella spp. involves the ability of the bacteria to penetrate and replicate within the epithelial cells of the large intestine. Model systems for examining the virulence of shigellae employ Henle intestinal epithelial cells in tissue culture and an in vivo assay for virulence in guinea pig eyes (Sereny test). Using these systems, we studied the genetic and physiological bases for the ability of shigellae to invade epithelial cells. We found that expression of virulence in Shigella spp. is dependent on the temperature at which the bacteria are grown. When grown at 37 degrees C, strains of Shigella flexneri 2a, Shigella sonnei, and Shigella dysenteriae 1 were fully virulent and invaded Henle cells. They also produced keratoconjunctivitis in guinea pigs. When grown at 30 degrees C, the bacteria neither penetrated Henle cells nor produced conjunctivitis in the Sereny test and were phenotypically avirulent. Strains grown at 33 degrees C were only partially invasive in the Henle assay, whereas strains grown at 35 degrees C were as invasive as strains grown at 37 degrees C. Using the Henle cell assay, we determined that the loss of ability to penetrate epithelial cells was completely reversed by shifting the growth temperature from 30 to 37 degrees C. The percentage of Henle cells invaded by bacteria increased with increasing time of growth at 37 degrees C. Restoration of invasiveness after growth at 30 degrees C required protein synthesis. When shigellae were grown at 30 degrees C and shifted to 37 degrees C for 2 h in the presence of chloramphenicol, the bacteria remained noninvasive. Similarly treated bacteria grown at 37 degrees C were still invasive. These results suggested that expression of one or more genes required for virulence of Shigella spp. are subject to regulation by growth temperature.     

Methods in laboratory investigation. Monoclonal antibodies to type IV collagen: probes for the study of structure and function of basement membranes

Type IV collagen is one of the main constituents of basement membranes, yet it is unknown whether the structural framework at different sites is assembled from one unique type of molecule or whether different type IV collagen molecules exist. To study the composition, chemical identity, and organization of this protein in different organs we have prepared monoclonal antibodies to a type IV collagen preparation from human placenta. Swiss Webster mice were hyperimmunized, and splenic cells were fused with the three different myeloma cell lines SP2/0, NS1, and U1. Type IV collagen-specific hybrids were selected and cloned by limiting dilution and on hard agar. Monoclonal antibodies secreted by two clones were extensively characterized by ELISA-inhibition assay, immunoprecipitation, rotary shadowing, and immunofluorescence techniques. Unlike conventionally raised antibodies in rabbits, both monoclonal antibody reagents show species-specific binding exclusively to native type IV collagen from human placenta but not to a similar preparation from calf lung or to other types of collagen. After heat denaturation of the antigen binding was no longer observed. The M3F7 antibody-binding site is located within the triple helical domain of the type IV molecule, approximately 900 A removed from the amino terminal end as visualized by a metal shadow casting technique. The monoclonal antibody M3F7 precipitates material from pepsin-derived and radiolabeled type IV collagen, and analysis of the polypeptide chains in the immunoprecipitate by sodium dodecyl sulfate polyacrylamide gel electrophoresis suggests that two major fragments are contained in the precipitate, which yield polypeptides of about 100 and 50 kilodaltons. After rotary shadowing of antigen-antibody mixtures native collagen fragments of two different size classes that bind antibody are visualized. One fragment is approximately 1500 A in length, and the other measures about 2700 to 3000 A. The localization of the antigenic site on these fragments suggests that both are generated by pepsin cleavage at a site about 900 A removed from the amino terminal end. In immunofluorescence experiments the monoclonal antibodies stained all basement membranes in kidney, lung, placenta, or skin, suggesting that at least the type IV collagen molecule recognized by these monoclonal antibodies is shared by a variety of vascular and epithelial basement membranes.     

Neutralizing antibodies to adenylate cyclase toxin promote phagocytosis of Bordetella pertussis by human neutrophils

A previous study showed that opsonization with human immune serum could either promote or antagonize phagocytosis of Bordetella pertussis by human neutrophils depending on whether the bacteria expressed adenylate cyclase toxin. Opsonization of the wild-type strain inhibited phagocytosis relative to unopsonized controls. In contrast, mutants lacking adenylate cyclase toxin were efficiently phagocytosed when opsonized with human immune serum. In this study, we examined opsonization in the presence or absence of monoclonal antibodies to adenylate cyclase toxin. Addition of neutralizing monoclonal antibodies to adenylate cyclase toxin converted a serum that previously inhibited both attachment and phagocytosis of the wild-type strain to one that increased both attachment and phagocytosis compared to the no-serum control. Monoclonal antibodies that recognize the adenylate cyclase toxin but fail to neutralize activity were without effect. These results suggest that adenylate cyclase toxin inhibits both Fc receptor-mediated attachment and phagocytosis of B. pertussis by neutrophils.     

Protective activities in mice of monoclonal antibodies against pertussis toxin.

Pertussis toxin (PT) protein, which is the most important protective antigen of Bordetella pertussis, has a hexameric structure composed of five subunits, designated S1 through S5. Immunoprotective activity of 20 different mouse monoclonal antibodies (MAbs) against pertussis toxin, 10 anti-S1, 1 anti-S2, 2 anti-S3, 4 anti-S23, and 3 anti-S4 antibodies, were investigated by aerosol and intracerebral challenges with virulent B. pertussis organisms in mice. Four anti-S1, named 1B7, 1D7, 3F11, and 10D6, and three anti-S23 antibodies, named 11E6, 10B5, and 10C9, showed the highest, and almost complete, protectivity against the aerosol challenge. Mouse protectivity against the intracerebral challenge was significant for these four anti-S1 MAbs but not for any of the three anti-S23 MAbs. Four anti-S1 and two anti-S4 MAbs did not protect the mice against either challenge. The other seven MAbs also showed dose-dependent moderate but significant protection against the aerosol challenge. In the aerosol challenge system, bacterial numbers and amounts of PT detected in the lung and the number of peripheral leukocytes were lower in the mice given the protective MAbs. All mice surviving 5 weeks after the infection produced high titers of antibodies against PT, filamentous hemagglutinin (FHA), and agglutinogens from the challenge organisms. A combination of the protective MAbs 1B7 and 11E6 strongly suppressed the disease and mortality of the mice at smaller amounts than with the anti-PT polyclonal antibody. Although combinations of one of the protective MAb and anti-FHA or anti-agglutinogen 2 also showed extremely high mouse protection without development of symptoms of the disease, antibody titers of the survivors against PT, FHA, and agglutinogens were significantly low. The foregoing results suggest that some important protective epitopes should be in S1 and S2 and/or S3, although there are both differences and similarities in the protective roles between anti-S1 and anti-S23 antibodies and also in the pathogenic mechanisms between aerosol and intracerebral infections. Furthermore, it was suggested that although not only FHA and agglutinogen 2 but also PT have roles as attachment factors, the processes of infection and protection are different between mice immunized with antibody against FHA or agglutinogen 2 and that against PT because the latter mice are also able to neutralize toxicity of PT diffused into the mice.     

Epitope mapping of monoclonal antibodies against Bordetella pertussis adenylate cyclase toxin

Adenylate cyclase (AC) toxin from Bordetella pertussis is a 177-kDa repeats-in-toxin (RTX) family protein that consists of four principal domains; the catalytic domain, the hydrophobic domain, the glycine/aspartate-rich repeat domain, and the secretion signal domain. Epitope mapping of 12 monoclonal antibodies (MAbs) directed against AC toxin was conducted to identify regions important for the functional activities of this toxin. A previously developed panel of in-frame deletion mutants of AC toxin was used to localize MAb-specific epitopes on the toxin. The epitopes of these 12 MAbs were located throughout the toxin molecule, recognizing all major domains. Two MAbs recognized a single epitope on the distal portion of the catalytic domain, two reacted with the C-terminal 217 amino acids, one bound to the hydrophobic domain, and one bound to either the hydrophobic domain or the functionally unidentified region adjacent to it. The remaining six MAbs recognized the glycine/aspartate-rich repeat region. To localize these six MAbs, different peptides derived from the repeat region were constructed. Two of the six MAbs appeared to react with the repetitive motif and exhibited cross-reactivity with Escherichia coli hemolysin. The remaining four MAbs appeared to interact with unique epitopes within the repeat region. To evaluate the roles of these epitopes on toxin function, each MAb was screened for its effect on intoxication (cyclic AMP accumulation) and hemolytic activity. The two MAbs recognizing the distal portion of the catalytic domain blocked intoxication of Jurkat cells by AC toxin but had no effect on hemolysis. On the other hand, a MAb directed against a portion of the repeat region caused partial inhibition of AC toxin-induced hemolysis without affecting intoxication. In addition, the MAb recognizing either the hydrophobic domain or the unidentified region adjacent to it inhibited both intoxication and hemolytic activity of AC toxin. These findings extend our understanding of the regions necessary for the complex events required for the biological activities of AC toxin and provide a set of reagents for further study of this novel virulence factor.     

Monoclonal antibodies to human endothelin-1: characterization and utilization in radioimmunoassay and immunocytochemistry.

A host of monoclonal antibodies directed against human endothelin-1 (ET-1) has been developed and characterized. The antibodies reacted with ET-1 specifically and with high affinity, as determined by competition analysis and sucrose density gradients. The antibodies did not cross-react with neuropeptide YY, beta-endorphin, calcitonin gene-related peptide, secretin or somatostatin. The antibodies cross-reacted with big endothelin (B-ET), endothelin-2 (ET-2), vasointestinal constrictor peptide (VIC), and endothelin-3 (ET-3) albeit with varying affinity but did not cross-react with sarafotoxin (SRTX-6b). None of the antibodies reacted with the C-terminal hexapeptide (HXPT) of ET-1, indicating that the epitopes are not located within this region of ET-1. The monoclonal antibodies exhibited binding activity in dilutions ranging from 1:1000, to 1:10(6). The isotypes of the monoclonal antibodies were determined by competition binding assay. Six of the monoclonal antibodies were of the IgG gamma 1, two were IgM and one of the IgG gamma 2a subclass. The antibodies detected immunoreactive ETs by radioimmunoassay and in immunocytochemical localization, suggesting the potential use of these antibodies as tools to determine the concentration of ETs in biological fluids and in immunocytochemical localization of ETs in specific cell types in various tissues.     

Monoclonal antibodies to human tumor necrosis factors alpha and beta: application for affinity purification, immunoassays, and as structural probes

Monoclonal antibodies were produced against two structurally related tumor necrosis factors (TNFs), TNF-alpha (previously called tumor necrosis factor) and TNF-beta (previously called lymphotoxin). The potential of these antibodies for the purification of TNFs, the development of specific immunoassays, and for defining the antigenic and functional domains of these cytokines was investigated. None of the monoclonal antibodies cross-reacted with both TNF-alpha and TNF-beta, or reacted with synthetic peptides which represented several of the regions of homology between these cytokines. Neutralizing monoclonal antibodies were utilized as immunoadsorbents to purify recombinant TNF-alpha and TNF-beta from E. coli lysates. TNFs purified by this method were greater than 98 percent pure by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and exhibited specific activities that were the same as TNFs isolated from natural sources using conventional chromatographic techniques. In addition, specific ELISA assays were developed that could detect less than 1 ng/ml of TNF-alpha or TNF-beta, and in contrast to bioassays, could discriminate between these related cytokines.     

Monoclonal antibodies to drugs--digoxin

A number of monoclonal antibodies to digoxin, two of which have been further characterized, were produced by somatic cell fusion. No cross reactivity with chemically related drugs was detected. Preliminary in vivo studies showed altered digoxin pharmacokinetics in antibody treated mice. These monoclonal antibodies could be used for a standardized radioimmunoassay and in the treatment of digoxin toxicity.