Immunoadsorbent Column Research Articles

Purification of polyamine oxidase from maize seedlings by immunoadsorbent column.

Plant polyamine oxidases (PAOs) are typical of the monocotyledones and are responsible for the terminal catabolism of polyamines, producing H2O2, diaminopropane and Δ′-pyrroline (from spermidine) or diazabicyclononane (from spermine)(Smith, 1985a; Smith et al., 1986). These enzymes are quite distinct from diamine oxidases (DAOs) which occur in the dicotyledones (Smith, 1985a; Rinaldi et al., 1986) the latter containing pyrroloquinolinequinone (Glatz et al., 1987; Suzuki 1987) as the cofactor while PAOs are flavoproteins (Smith, 1983). In general DAOs have a broader substrate specificity oxidizing diamines but also spermine and spermidine (Chaudhuri and Gosh, 1984).KeywordsWater HyacinthMaize SeedlingDiamine OxidasePolyamine OxidaseJerusalem Artichoke TuberThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Use of immunoadsorbent columns for antiacetylcholine receptor antibody removal from plasma of myasthenia gravis patients

The best apheretic approach to myasthenia gravis is the selective removal of antiacetylcholine receptor antibodies (antiAchR-Ab) from plasma. A new tryptophan-linked polyvinyl-alcohol gel recently reported to be able to adsorb IgG autoantibodies semiselectively from plasma was investigated in vitro. A consistent reduction of antiAchR-Ab ranging from 76% to 100% was observed in all plasma samples tested. Various degrees of reduction of other immunoglobulins were noted. The regeneration of the immunocolumn did not reduce the efficiency in autoantibody removal. Further in vitro and in vivo investigations are needed to confirm the clinical usefulness of this promising apheretic approach to myasthenia gravis.

Ultraviolet-induced suppressor T cells and factor(s) in murine contact photosensitivity

Murine contact photosensitivity (CPS) to 3,3',4',5-tetrachlorosalicylanilide (TCSA) is a highly specific, T-cell-mediated delayed-type hypersensitivity (DTH). Preexposure of the photosensitizing site to low doses of ultraviolet B(UVB) rendered mice unresponsive to challenge reaction. This unresponsiveness was associated with the generation of antigen-specific, afferent limb-acting, Lyt-1+2-,L3T4+ suppressor T cells (Ts-cps) in the spleen, thymus, and lymph node. Cell-free extract(s) obtained by freezing and thawing of these cells contained T-cell-suppressor factor (TsF) that inhibited the development of the induction phase of the CPS response to TCSA in vivo in an antigen-specific fashion. The treatments of TsF both with immunoadsorbent columns and with reduction and alkylation showed that the factor bore photoantigen-binding site(s), was reactive with monoclonal anti-I-Jd, anti-I-E alpha but not anti-I-Ad, and behaved as a single-chain factor containing both photoantigen binding and I-J molecules. By gel chromatography the majority of the suppressive activity was eluted in the fractions corresponding to molecular weights of 60-80 and 100-200 kDa. Our present study demonstrated clearly that UVB-induced unresponsiveness in the DTH reaction was mediated by a soluble suppressive factor derived from T cells.

Electrophoretic Elution from Monoclonal Antibody Immunoadsorbents: A Theoretical and Experimental Investigation of Controlling Parameters

AbstractAn experimental and theoretical investigation of the mechanism of electrophoretic elution is described. The rate of elution of bovine serum albumin from monoclonal antibody immunoadsorbents was observed to increase with increasing electric field, increasing fractional saturation, and decreasing height of the immunoadsorbent column. Slower elution rates were observed from immunoadsorbents possessing higher affinities and capacities. A reaction‐migration mechanism is proposed in which the packed bed is modeled as a continuous hydrogel and in which the electrical field causes antigen that has spontaneously dissociated to migrate through the column under conditions of bound‐free equilibrium. Good qualitative and quantitative agreement is obtained between experiment and the predictions of a numerical model. A membrane device suitable for large‐scale application of this technique is discussed.

DERMAL REACTIVITY TO HUMAN CYTOMEGALOVIRUS (CMV)

A skin test for immunity to human CMV is described in which skin induration is measured after intradermal injection of heat-inactivated Towne strain CMV antigen. Randomly selected healthy young adult males and non-pregnant females were pre-screened for evidence of past infection with CMV using a latex agglutination test. Each individual was inoculated with 100 ul of test antigen (50 ug) prepared from serum-free supernatants of CMV-infected MRC-5 cells (inactivated at 56°C for 6 hrs). Candida extract (1:1000) and non-infected MRC-5 cell lysates. CMV seropositive individuals elicited positive skin reactions to both the Candida extract and the test antigen. No response was observed at the MRC-5 cell lysate inoculation site. Seronegative individuals who were negative to the test antigen at the start of the study, developed a positive response 8 days after intramuscular immunization with live attenuated Towne strain CMV. This response also correlated with in vitro CMV- induced lymphocyte proliferation of peripheral blood lymphocytes from the immunized individuals and persisted for at least 93 days. In guinea pig experiments with human CMV, those animals immunized with purified CMV, a virus envelope preparation, or a 130-55K glycoprotein complex (isolated by immunoadsorbent column chromatography), developed strong skin reactions to intradermal injection of 5 ug of each of these antigens. A weak reaction was also observed to viral capsid antigen. No reactions were observed in non-immune animals.

Affinity purification of tetanus toxin using polyclonal and monoclonal antibody immunoadsorbents.

Tetanus toxin has been immunopurified on immunoadsorbent columns derived from equine polyclonal antitoxin coupled to cyanogen bromide-activated Sepharose CL4B. Desorption of bound toxin in active form was achieved only when the immunoadsorbent was mixed with Sephadex G15 and this mixture overlaid on a further volume of Sephadex G15. With equine antibody, 64% of adsorbed toxin was recovered with a specific activity of 2400 limiting flocculation units (Lf)/mg protein N (1.2 X 10(8) minimum lethal doses (MLD)/mg protein N). Similarly prepared immunoadsorbent derived from murine monoclonal antitoxin of low affinity had improved desorption with less acidic desorbents, without the requirement for Sephadex G15; greater than 80% of adsorbed toxin was recovered with a specific activity of 3000 Lf/mg protein N (1.6 X 10(8) MLD/mg protein N).

Antigen-specific suppression of the in vitro cytotoxic response by a soluble factor produced by corynebacterium parvum-induced allospecific suppressor cells

The adjuvant Corynebacterium parvum, when administered intravenously during an ongoing alloimmunization, induces alloantigen-specific splenic suppressor cells which inhibit primary and secondary in vitro sensitizations. We have previously shown that these cells produce a soluble suppressor factor in culture. We now further characterize this factor and its mechanism of action. Release of this suppressive factor is dependent upon specific restimulation of the splenic suppressor cell with the sensitizing alloantigen for 24–48 hr in culture. The suppressor factor inhibits primary, but not secondary, in vitro sensitizations in an antigen-specific, genetically unrestricted manner. The suppressive activity is not absorbed by passage through immunoadsorbent columns containing anti-mouse immunoglobulin. The factor does not lyse tumor cells bearing the sensitizing alloantigen. Delay in addition to primary cultures of as little as 4 hr after culture initiation leads to loss of suppressive activity, suggesting that this antigen-specific allosuppressor factor inhibits an early step in the sensitization of precursor cytotoxic T lymphocytes.

29] Isolation of human erythropoietin with monoclonal antibodies

Human erythropoietin was isolated from urine of aplastic anemic patients in a high yield with a simple purification procedure using an immunoadsorbent column of monoclonal antibodies and a Sephadex G-100 column. About 6 mg of erythropoietin was isolated from 700 liters of urine and the specific activity was estimated to be 81,600 units/mg of protein with an in vivo 59Fe incorporation assay method, using starved rats. Activity measurement of the extracts from sliced gels after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the Western blotting technique revealed heterogeneity of the isolated erythropoietin, which is probably caused by variable amounts of carbohydrates attached to the polypeptide chain. Thirty amino acids in the NH2-terminal portion of the isolated hormone were sequenced.

Similarity among Organ Region Protein Antigens of Pea

Summary Immunodiffusion, two-dimensional crossed rocket immunoelectrophoresis and immunoadsorbent column chromatography were used in an attempt to isolate an organ region-specific antibiody against organ region total protein extracts of pea. Although organ region-specific differences could by detected by immunodiffusion, it was not found possible to isolate an organ region-specific antibody.

Monoclonal antibody to noncatalytic subunit of bovine plasma factor XIII : Selective isolation of catalytic subunit.

Plasma Factor XIII is a zymogen of plasma transglutaminase (EC 2.3.2.13), which is responsible for the intermolecular crosslinking of fibrin molecules during blood clotting. The factor is a tetramer (a2b2) comprising catalytic (a2) and noncatalytic (b2) subunits. By fusing mouse myeloma cells with spleen cells of a mouse immunized with Factor XIII protein that was partly purified from bovine plasma, we generated a hybridoma clone (A2) that synthesized a monoclonal antibody against bovine plasma Factor XIII. The subclass of IgG produced by clone A2 was IgG2a. The monoclonal antibody was not cross-reactive with human placental Factor XIII or guinea pig liver transglutaminase. Experiments with a monoclonal-antibody immunoadsorbent column indicated that the monoclonal antibody recognized an epitope on the noncatalytic subunit, and that bovine plasma Factor XIII also dissociated into catalytic and noncatalytic subunits in the presence of a high concentration of Ca2 +, as human plasma Factor XIII does. The cata...

Immunization of DBA/2 mice with a T cell hybridoma-derived TsF increases immune resistance to the syngeneic tumors P815 and L1210.

The ability of a tumor-specific T suppressor factor (TsF) isolated from a T cell hybridoma, A10, to act as an immunogen in DBA/2 mice was investigated. The TsF was affinity purified from ascites over an immunoadsorbent column containing a monoclonal antibody (B16G) that has specificity for the TsF molecule, or over columns containing membrane extracts of the P815 mastocytoma (the tumor for which A10 is specific). The specificity control was BW5147 (the fusion partner for A10) membrane extracts treated in the same way as A10. DBA/2 mice were immunized with the affinity-purified material or PBS and were subsequently challenged with either the P815 tumor or the L1210 DBA/2 thymoma. When mice were immunized with material affinity purified over B16G, eluted material from both A10 ascites and BW5147 membrane extracts enhanced resistance to both P815 and L1210 challenge, indicating that B16G was binding immunogenic material derived from both preparations, which exerted a tumor-protective effect. However, when a P815 affinity column was used, protective material was eluted only from A10 ascites, and this bestowed resistance to both P815 and L1210. When irradiated whole cells were used as immunogens, only A10 cells stimulated anti-tumor immunity, and this appeared to be directed specifically to the P815 tumor. The implications of these findings in terms of the potential for immune modulation with anti-suppressor therapy, and the specificity of the B16G monoclonal, are discussed. The demonstration of B16G binding material (TsF) in the membranes (but not the ascites) of the BW5147 line is also of significance to investigators using BW5147 fused suppressor hybridomas.

Humoral antibody response against Bacteroides gingivalis-specific antigen recognized by monoclonal antibody in adult periodontal patients

An antigen recognized by monoclonal antibody specific for Bacteroides gingivalis was purified in the presence of 0.5% (wt/vol) beta-octyl-glucoside by immunoadsorbent column chromatography. The purified antigen was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining, and the pattern of SDS-PAGE agreed with that of immunoblotting. The molecule exhibited an apparent molecular weight of about 62,000 by SDS-PAGE. The antigen was sensitive to trypsin, Staphylococcus aureus V8 protease, DNase I and II, and heating, but insensitive to RNase and neuraminidase. By the enzyme-linked immunosorbent assay method, the purified antigen was not cross-reactive with rat polyclonal antibodies to each of several black-pigmented Bacteroides species, Fusobacterium nucleatum, Eikenella corrodens, and Actinobacillus actinomycetemcomitans. These results indicate that the purified antigen is specific for B. gingivalis. Humoral antibody titers in adult periodontal patients against the specific antigen were measured by the enzyme-linked immunosorbent assay. Serum immunoglobulin G antibody titers against the specific antigen in adult periodontal patients correlated significantly with the severity of periodontal disease. However, such significant correlation was not observed with serum immunoglobulin M antibody titers.

Indirect immunoselection of late distal cell populations from rabbit kidney cortex.

This study describes a method for the separation of distal cell populations based on the sequestration of proximal cells on immunoadsorbent columns (CNBr-activated Sepharose 6MB) bound with three brush-border monoclonal antibodies (S6-Mab). A high yield of isolated cell suspension from rabbit kidney cortex was prepared by mechanical dissociation after perfusion and incubation of the kidneys with 10(-3) M EDTA. The sequestration of the proximal cells was achieved in two sequential chromatographic steps. About 92% of the applied cells were first retained on an S6-Mab column after a 60-min stationary stage and the unbound cells were submitted by direct flow to a second S6-Mab column. In such conditions, 8 X 10(6) cells were recovered when starting with 331 X 10(6) cortical cells. The efficiency of the proximal cell depletion process was confirmed by an 80% decrease in brush-border enzymes, a very low phosphoenolpyruvate carboxykinase activity, and absence of cells bearing long microvilli, as ascertained by electron microscopy. This immunodepleted cell population presented the enzymatical characteristics of cells from the more distal segments. As compared with the initial cell suspension, these cells exhibited higher hexokinase (2.3 times), succinate dehydrogenase (1.5 times), and Na+-K+-ATPase (2.6 times) activities. In addition, adenylate cyclase activities remained sensitive to parathormone, arginine vasopressin, and isoproterenol. The functional capacity of these immunodepleted cells was assessed by an almost complete exclusion of eosin dye, a low Na+ and high K+ intracellular content, and a high respiratory rate of oxygen consumption. In conclusion, this immunoselective process makes it possible to obtain subpopulations of renal cortical cells possessing the main characteristics of the distal, connecting, and collecting cells for physiological and metabolic studies.

Synergy between recombinant interleukin 2 (rIL 2) and IL 2-depleted lymphokine-containing supernatants in facilitating allogeneic human cytolytic T lymphocyte responses in vitro.

Supernatants from human mixed leukocyte cultures or lectin-depleted supernatants from cultures of PHA-activated human peripheral blood leukocytes were depleted of IL 2 by passage over an anti-human rIL2 immunoadsorbent column. The column eluates were concentrated, dialyzed, and tested for their ability to synergize with human rIL 2 in facilitating human cytolytic T lymphocyte (CTL) responses to allogeneic, uv-irradiated HT144 melanoma cells in vitro. CTL were generated in the presence of 1 X 10(-4) M hydrocortisone sodium succinate in order to minimize the generation of nonspecific lymphokine-activated killer (LAK) cells. IL 2-depleted lymphokine-containing supernatant (LKS), alone or in the presence of less than or equal to U/ml rIL 2 did not stimulate significant CTL responses. Recombinant IL 2 at greater than 2 U/ml stimulated weak CTL responses in the absence of LKS. However, strong synergistic CTL responses were observed when both IL 2-depleted LKS and greater than 2 U/ml rIL 2 were added to the cultures. CTL generated in these cultures could be distinguished from nonspecific LAK cells on the basis of their i) specificity, ii) T3 phenotype, and iii) kinetics of generation. Nevertheless, rIL 2 and IL 2-depleted LKS were sometimes observed to synergize in facilitating the generation of nonspecific LAK cells as well as the generation of specific CTL. When the times at which rIL 2 and IL 2-depleted LKS were added to the cultures were varied, IL 2 was found to be required early in CTL responses, whereas the synergistic factor(s) in LKS seemed to act later. Recombinant human interferon-gamma was unable to replace LKS in synergizing with rIL 2 to elicit CTL responses. In summary, these experiments suggest that LKS contains a late-acting factor(s), antigenically distinct from IL 2, which synergizes with IL 2 in facilitating human CTL responses.

Immunochemical characterization of the cell surface carbohydrate antigens of Peptostreptococcus anaerobius.

Two carbohydrate antigens were isolated from the cell surface of Peptostreptococcus anaerobius. One, extracted from purified cell walls with NaOH, contained glucose and phosphorus, with traces of galactosamine and glucosamine. Serological activity was detected by a 'dot blot' procedure. The second antigen, extracted from cell membranes with phenol and purified by chromatography on Sepharose 6B and an immunoadsorbent column, contained glucose, glycerol phosphate, phosphorus and fatty acids. Antigenicity of this extract could also be demonstrated by an ELISA technique.

Characterization of a monoclonal antibody directed against the biologically active site of human interleukin 1.

Human IL-1 was successfully used to produce an anti-IL-1 mAb. Anti-IL-1 (IgG2a) blocked IL-1-mediated thymocyte and fibroblast proliferation, but did not interfere with the biological effects of other lymphokines, such as IL-2 or IL-3. The antibody immunoprecipitated biosynthetically radiolabeled 33, 17, and 4 kD IL-1. An immunoadsorbent column yielded 20% of initial activity, and upon HPLC size-exclusion chromatography, affinity-purified IL-1 had a molecular mass of approximately 4 kD. These results provide first evidence of a monoclonal anti-IL-1 that reacts with different species of IL-1 and apparently binds to an epitope close to the active site of IL-1. Thus, anti-IL-1 IgG may be very helpful for further investigations of the molecular as well as biological characteristics of IL-1 and related mediators.

Isolation of epidermal growth factor with monoclonal antibody.

Three kinds of monoclonal antibodies directed to human epidermal growth factor (hEGF) were obtained. One of them did not react with mouse epidermal growth factor (mEGF) but the others showed reactivity with mEGF. Antibody having binding ability to both hEGF and mEGF was suitable for the purification of EGFs. Human EGF was isolated from normal adult human urine in a high yield by means of a simple purification process using an immunoadsorbent column of monoclonal antibody. Mouse EGF was also purified from mouse submaxillary gland using the same immunoadsorbent column and a Sephadex G-50 column. Both of the purified EGFs gave a single band with an apparent molecular weight of 6000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The amino acid sequences were consistent with those reported previously.

28] Cytotoxicity by natural killer cells: Analysis of large granular lymphocytes

This chapter focuses on the purification of large granular lymphocytes (LGL) cells and their subpopulations with the use of a variety of classical separation procedures as well as monoclonal antibodies. The cytotoxic and noncytotoxic functions of LGL are also described in the chapter. Natural killer cells (NK) are effector cells with spontaneous cytotoxicity against target cells, which lack the properties of classical macrophages, granulocytes, or cytotoxic T cells. NK cells have been primarily defined by their ability to lyse target cells in vitro. Moreover, it is necessary to purify these cells to adequately assess their ability to mediate cytolysis as well as other noncytolytic functions and to characterize them in regard to their biological role in vitro and in vivo. LGL subsets are separated on an anti-F(ab′)2 immunoadsorbent column. Binding efficiency of the protein ranges from 40 to 70% as determined by measuring the reduction in the optical density at 280 nm of the protein solution after the coupling.

Monoclonal antibodies to avian lipoprotein lipase. Purification of the enzyme by immunoaffinity chromatography

Three monoclonal antibodies to avian lipoprotein lipase have been isolated by fusing spleen cells from immunized BALB/c mice with myeloma P3X-63 Ag 8. The antibodies were detected by their ability to bind immobilized lipoprotein lipase in enzyme-linked immunosorbent assay (ELISA) and by immunoprecipitation of purified enzyme in the presence of second (rabbit anti-mouse) antibodies. Two of these antibodies, CAL1-7 and CAL1-11, inhibited catalytic activity, whereas with CAL1-2 interaction with lipoprotein lipase could be demonstrated only in ELISA and in Western blot assays following denaturation of the enzyme with sodium dodecyl sulfate. An immunoadsorbent column was prepared by coupling immunopurified CAL1-11 to Sepharose-4B. When acetone powder extracts of adipose tissue were applied on the column, 70% of the catalytic activity bound to the matrix. Effective elution was achieved with 1.8 M NaCl, 40% glycerol, 5% acetone, 20 mM Chaps (3[(3-holamidopropyl)dimethylammonio]propanesulfonate), 0.5 mM EDTA, 1 mM phosphate (pH 6.5). After concentration of the active fractions on a heparin-Sepharose 4B column, the purified enzyme was obtained with an overall recovery of 25%. Sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrates that the preparation is homogeneous with a major band at M r 60900. Thus, avian adipose lipoprotein lipase has been purified by a one-step immunoaffinity followed by a concentrating step on heparin-Sepharose 4B.

Production of auto-anti-idiotypic antibody during the normal immune response: VIII. Effect of auto-anti-idiotypic antibody on contact sensitivity

An eluate prepared by brief incubation of spleen cells from 2,4,6-trinitrophenyl (TNP) lysyl-Ficoll immunized mice with TNP-epsilon-amino-n-caproic acid causes a specific inhibition of the induction of contact sensitivity by 2,4,6-trinitrochlorobenzene skin painting. The active factor in the eluate binds to an anti-mouse immunoglobulin (Ig) immunoadsorbent column but not to a TNP immunoadsorbent column and is therefore Ig but not anti-TNP antibody. The active factor does bind to an immunoadsorbent prepared from anti-TNP antibody, suggesting that the factor has anti-idiotype specificity. Evidence based upon hapten-reversible inhibition of plaque formation and an enzyme-linked immunosorbent assay (ELISA) indicates that the eluates contain auto-anti-idiotype antibody specific for anti-TNP antibody. It is suggested that auto-anti-idiotype antibody spontaneously produced during the immune response to a T-independent antigen can specifically downregulate contact sensitization to the same epitope.