Simple procedure for purification of diphtheria toxin and separation of its fragments by hydrophobic chromatography

Diphtheria toxin and fragment B bind to hydrocarbon-coated agaroses. Fragment A of the toxin is not adsorbed to such resins. Using Seph-C₄, the toxin and fragment B can be eluted from the column after adsorption by increasing the ionic strength of the eluent. The toxin is also eluted from the Seph-C₆ column, but fragment B is eluted only in the denatured form. Purification of the toxin can be achieved simply by passing the growth medium supernatant through a small size Seph-C₆ column and eluting the toxin by 0.1 m NaCl. The fragments of diphtheria toxin obtained after mild trypsin treatment can be separated purely on a Seph-C₄ column. The hydrophobic chromatography system may thus serve as a tool for purification of the toxin and its fragments: it may also be useful in large-scale preparations.     

Hydrophobic chromatography in the purification of the histidine-binding protein J from Salmonella typhimurium

Upon increasing the length of the side chains in a homologous series of ω-aminoalkyl agaroses (Seph—C_n—NH₂), more and more proteins become adsorbed onto the column and it is thus possible to achieve purification by selective exclusion of a desired protein. This principle is illustrated in the purification of the histidine-binding protein j from Salmonella typhimurium, vising ω-aminodecyl or ω-aminododecyl agarose columns. The protein was purified 7- to 10-fold in one step from either the shock fluid or the crude extract of the bacteria.Since protein j has an isoelectric pH of 5.5 and yet does not bind to the positively charged columns at pH 7, it seems that this protein is not likely to have available hydrophobic pockets and may thus be remarkably hydrophilic, compared with the other proteins in the shock fluid.     

Immunoglobulin Preparations Can Mislead Clinical Decision-Making in Follow-Up of Differentiated Thyroid Cancer

Objective: Intravenous and subcutaneous immunoglobulins are commonly used for immune substitution or as immune modulators in a variety of inflammatory and autoimmune disorders. Exogenous thyroid-specific thyroglobulin (Tg) antibodies present in the donor plasma may interfere with the interpretation of measurements of Tg autoantibodies (Tg-Abs) in the recipient’s plasma and potentially trigger an immune response in the recipient’s immune cells. Levels of antibodies causing bioassay interferences or those leading to clinically relevant changes in patient outcomes are not known. Tg is used as a biomarker in the long-term surveillance of patients with differentiated thyroid cancer (DTC) following total thyroidectomy and radioactive iodine ablation. However, the presence of Tg-Abs in the circulation interferes with Tg measurements. Assessment of levels of Tg-Abs is thus recommended as a part of standard follow-up of DTC together with Tg testing.Methods: To understand the potential mechanisms and pathophysiologic significance of possible interferences associated with administration immunoglobulin preparations and Tg measurement, we overview the current knowledge on interactions between Tg autoimmunity and immunoglobulin preparations and illustrate diagnostic challenges and perspectives for follow-up of patients with DTC treated with exogenous immunoglobulins.Results: In patients with DTC treated with immunoglobulin preparations, monitoring of thyroid cancer using Tg and Tg-Abs is challenging due to possible analytical interferences through passive transfer of exogenous antibodies from immunoglobulin preparations.Conclusion: Analytical interferences must be suspected when a discrepancy exists between clinical examination and diagnostic tests. Collaboration between endocrinologists, biologists, and pharmacologists is fundamental to avoid misdiagnosis and unnecessary medical or radiologic procedures.Abbreviations: CT = computed tomography; DTC = differentiated thyroid cancer; FNAB = fine-needle aspiration biopsy; HAb = heterophile antibody; IMA = immunometric assay; IVIg = intravenous immunoglobulin; RAI = radioactive iodine; RIA = radioimmunoassay; SCIg = subcutaneous immunoglobulin; Tg = thyroglobulin; Tg-Ab = thyroglobulin autoantibody; Tg-MS = thyroglobulin mass spectrometry; TPO-Ab = thyroid peroxidase autoantibody; TSHR-Ab = thyrotropin receptor autoantibody     

Use of monoclonal antibodies against horse immunoglobulin in an enzyme immunoassay of bacterial toxins and toxoids

Immunization of BALB/c mice by horse antiserum against diphtheria made it possible to obtain IgG₁ monoclonal antibodies (MoAbs) 2B7E4 specific for light chains of horse immunoglobulin (Ig). Unlike commercial preparations of anti-horse immunoglobulin antibodies, which are specific for the whole Ig molecule or its Fc-fragment, the peroxidase (HRP) conjugate of the MoAb, 2B7E4-HRP did not interact with human, mouse, rabbit, and sheep Igs, or horse albumin. The conjugate obtained was used with MoAbs against bacterial toxins and commercial horse antitoxins, as a universal reagent in sandwich enzyme immunoassay (ELISA) for bacterial toxins and toxoids. The detection sensitivity of diphtheria toxin/toxoid equaled 0.0005 Lf/ml; tetanus toxin and toxoid were detected with sensitivities of 20 LD₅₀/ml and 0.005 UI/ml, respectively. A similar sandwich ELISA for botulinum toxoids (group measurement) allowed types A, B, and E to be detected at 0.02, 0.002, and 0.001 UI/ml, respectively; selective measurement was only possible in the case of type E toxoid (0.001 UI/ml).     

Production and Characterization of Monoclonal Antibodies to Wall-Localized Peroxidases from Corn Seedlings

A library of 22 hybridomas, which make antibodies to soluble wall antigens from the coleoptiles and primary leaves of etiolated corn (Zea mays L.) seedlings, was raised and cloned three times by limit dilution to assure monoclonal growth and stability. Two of these hybridomas made immunoglobulin G antibodies, designated mWP3 and mWP19, which both effectively immunoprecipitated peroxidase activity from crude and partially purified preparations of wall peroxidases. Direct peroxidase-binding assays revealed that both antibodies bound enzymes with peroxidase activity. As judged by immunoblot analyses, mWP3 recognized a M_r 98,000 wall peroxidase with an isoelectric point near 4.2, and mWP19 recognized a M_r 58,000 wall peroxidase. Immunogold localization studies showed both peroxidases are predominately in cell walls.     

Phosphorylation of the IgE receptor from ionophore A23187 stimulated intact rat mast cells

Purified rat serosal mast cells were labeled either with [³²P]orthophosphate or [³⁵S]methionine and their receptors for immunoglobulin E were isolated by repetitive affinity chromatography. In ³⁵S-labeled receptor preparations SDS polyacrylamide gels revealed a broad receptor band, M_r 45,000 to 53,000, and two other bands, M_r 30,000 and 16,000, which apparently represent receptor-associated proteins. Only the receptor band was labeled by ³²P. Phosphorylation of receptor was markedly stimulated by the divalent cation ionophore A23187, a known stimulator of histamine release, with changes occuring as early as 15 seconds. This early increase in receptor phosphorylation may be involved in the control of mediator secretion.     

Cotranslational cleavage of immunoglobulin light chain precursors by plasmacytoma microsomes.

Murine plasmacytoma endoplasmic reticulum which has been freed of ribosomes by EDTA treatment is capable of the cotranslational proteolytic processing of representative λ₁,λ₂, and k immunoglobulin light chain precursors. Messenger RNA fractions from the MOPC-104E, MOPC-315, and MOPC-46B tumor lines were used to direct the synthesis of the light chain precursors in a cell-free system derived from Krebs II ascites cells. The precursor cleavage activity of the plasmacytoma membranes is comparable in activity and in characteristics to that of two well-defined membrane preparations: Krebs II ascites intracellular membranes (E. Szczesna and I. Boime, 1976, Proc. Nat. Acad. Sci. USA73, 1179–1183) and EDTA-treated rough endoplasmic reticulum from canine pancreas (34., 35., J. Cell Biol.67, 852–862). The efficiency of the cleavage reaction appears to be dependent upon the precursor being utilized as a substrate. An assay suitable for a preliminary characterization of the plasmacytoma membrane preparations is described.     

Protein binding to two-dimensional hydrophobic binding-site lattices: adsorption hysteresis on immobilized butyl-residues

Long-lived metastable states involving multiple binding sites of a protein ligand with immobilized alkyl residues on a solid phase can be observed at high ionic strength between butyl agaroses (5.21 μ mol/ml packed gel) and phosphorylase b by perturbations enforcing either the on-reaction (adsorption) or the off-reaction (desorption). These apparent equilibrium states are suggested because the adsorption isotherms of phosphorylase b on butyl agaroses are not retraced by the desorption isotherms. In this first example of macromolecular adsorption hysteresis on immobilized alkyl residues, it can be shown that the irreversible entropy (Δ_iS) produced in an adsorption-desorption cycle lies between 6 (5 μ mol/ml packed gel) and 40 (21 μ mol/ml packed gel) J mol ¹ K⁻¹. For the latter gel the apparent standard entropy of adsorption (Δ_aS_i⁰′) is 160 J mol⁻¹ K⁻¹. The metastable state observed during adsorption is probably due to an energy barrier which must be overcome for the nucleation of protein binding on the matrix. Other metastable states may possibly be encountered during desorption when the adsorbed enzyme resists the breakage of hydrophobic interactions. In the transition from the adsorption branch to the desorption branch of the hysteresis loop, the apparent affinity of the enzyme-matrix interaction is enhanced. For the desorption branch, the apparent association constant of half-maximal saturation corresponds to K_d,0.5′ = 4.2 × 10⁹ ]m⁻¹ as compared to the respective constant of adsorption K_{a, 0.5}′ = 1.6 × 10⁵m⁻¹ (gel: 21μ mol/ml packed gel). Since the area of the hysteresis loops (see also Δ_iS) depends strongly on the density of butyl residues on the gel, it is concluded that the number of alkyl residues interacting with the protein molecule is crucial for the metastable states and hysteresis. It is unlikely that hysteresis is due to the pore structure of the agarose or to nearest neighbour interactions of ligand molecules. Since thermodynamic irreversibility and hysteresis may be encountered when macromolecules, such as proteins, are adsorbed to cell membranes or cell organelles: an analysis and understanding of these phenomena should be of general biological significance.     

Preparation and characterization of antibody to galactosyl(α1 → 4)galactosyl(β1 → 4)glucosylceramide

Antiserum against galactosyl(α1 → 4)galactosyl(β1 → 4)glucosylceramide (globotriaosylceramide, Gb₃) was raised in rabbits by the administration of four weekly intramuscular injections of 1.5 mg of the purified glycolipid along with bovine serum albumin and Freund's complete adjuvant. AntiGb₃ activity was quantitated initially by immunoprecipitation employing Gb₃ mixed with 100-fold excess of lecithin and cholesterol (1 : 1 or 1 : 2, by wt.) as antigen. Subsequently, complement fixation tests done with antigen preparations containing Gb₃/lecithin/cholesterol (1 : 6 : 20, by wt.) showed antiGb₃ titres of up to 1 : 8192. Fractionation of the antiserum by BioGel A5m chromatography indicated the antibody was an IgM immunoglobulin. The partially purified antibody stimulated complement-dependent release of glucose from glucose-containing liposomes prepared with sphingomyelin/cholesterol/dicetylphosphate/Gb₃ (molar ratio, 100 : 75 : 11 : 1). The antibody crossreacted with the trisaccharide, Gal(α1 → 4)Gal(β1 → 4)Glc, but not with galactosylceramide, lactosylceramide, GM1 ganglioside, globotetraosylceramide, digalactosyldiglyceride or a number of low molecular weight saccharides.     

Relaxation of isolated human pulmonary muscle preparations with prostacyclin (PGI2) and its analogs

The effects of PGI₂ and two analogs Iloprost and ZK 96480 were examined on isolated human pulmonary muscle preparations. High concentrations of these agents reduced the basal tone in all types of preparations. In addition, they relaxed tissues which had been maximally contracted with histamine (50 μM). PGI₂ was more potent on pulmonary arterial muscle preparations (pD₂ value : 6.33, n = 3) than on bronchial muscles. The relaxations induced by PGI₂ in bronchial preparations were quite variable, that is, some tissues relaxed while others did not. The analogs also relaxed arterial preparations and the pD₂ values were approximately the same (Iloprost : 7.42, n = 4 and ZK 96480 : 7.48, n = 4). The isolated human pulmonary vascular preparations were approximately 10-fold more sensitive to the analogs than bronchial muscle preparations. In bronchial tissues we noted that the PGI₂ relaxant effect was spontaneously reversed with time, an activity not observed with both analogs. A pretreatment of the bronchial tissues with indomethacin (1.7 μM) did not reduce the variations observed with PGI₂ nor modify the transient relaxation observed with this agent. These data demonstrate that vascular tissues from the human lung are considerably more sensitive to these relaxant agonists than bronchial preparations.     

Studies on the cationization of agarose

Cationized agaroses with different degrees of substitution (0.04–0.77) were synthesized, employing 3-chloro-2-hydroxypropyltrimethylammonium chloride (CHPTAC). The influence of different reaction parameters on the substitution degree and molecular weight was evaluated. The investigated parameters were concentration of reagents, temperature, time, and addition of NaBH₄. The products were characterized by means of scanning electronic microscopy, infrared spectroscopy, viscosimetry, and NMR spectroscopy. Methanolysis products were studied by electrospray ionization mass spectrometry. The higher the concentration of CHPTAC employed, a higher degree of substitution was obtained, if the optimum concentration of NaOH in each case was employed. Insufficient quantities of NaOH reduced epoxide formation and the reacting alkoxides of the polysaccharide, whereas an excess of NaOH favored degradation of the epoxide and decrease in the molecular weight of the product. A reaction time of 2 h was sufficient to obtain products with the maximum degree of substitution for each case. The addition of NaBH₄ gave products with a slightly higher molecular weight, but the extra cost involved should not justify its use for large-scale application.     

Isolation and characterization of plasma membrane associated immunoglobulin from cultured human diploid lymphocytes

The lactoperoxidase iodination method was adapted to label surface proteins of cultured diploid human lymphocytes. Membrane associated immunoglobulin of the μ,K type was isolated from WIL₂-A3 cells as well as from their purified membrane preparations by detergent solubilization of labeled membrane proteins and subsequent precipitation with specific antisera. These data indicate that using our conditions all of the labeled immunoglobulin was membrane bound. The molecular weight of the bound molecule was estimated to be 265,000±15,800 by sodium dodecyl sulfate gel electrophoresis and on reduction was separated into proteins with molecular sizes identical to μ and light-chain markers. The combination of two μ and two light chains to give an “IgM monomer” configuration should give a molecular weight of 180,000 to 200,000. Possible reasons for this discrepancy are discussed.     

Functional Activities of Antibodies Directed against Surface Lipoproteins of Borrelia hermsii

Enriched preparations for mouse polyclonal immunoglobulin G (IgG) antibodies reactive with surface-exposed epitopes (Ab-SEE) of the 22-kDa and 24-kDa membrane lipoproteins of living Borrelia hermsii (HS 1 strain) cells were obtained by an antibody absorption technique using living spirochetes. In vitro, the antibody preparations both inhibited spirochetal growth and were borreliacidal in the presence of complement. The monovalent Fab antibody fragments, prepared from antibody-enriched preparations, did not inhibit the growth of the bacteria, whereas they killed the bacteria in the presence of complement. The two-dimension gel electrophoresis of B. hermsii cells showed that ³H-labeled fatty acids incorporated into the 22-kDa and 24-kDa lipoproteins were resolved into one and three compact spots, respectively. The spots were recognized by the Ab-SEE preparations reactive with the 22-kDa and 24-kDa proteins, by Western blotting.     

Identification of solubilised beta1 and beta2 adrenoceptors in mammalian lung

The properties of beta-adrenoceptors in solubilised and particulate preparations of rat and rabbit lung have been assessed using the specific ligand (³H)-dihydroalprenolol ((³H)-DHA). Membranes were solubilised using the detergent digitonin and the specific binding of (³H)-DHA assayed using a charcoal-centrifugation technique to separate free and bound ligand. The equilibrium dissociation constant (K_D) of specific (³H)-DHA binding was very similar in particulate and soluble preparations of rat and rabbit lung. Moreover, the optical isomers of propranolol displayed virtually identical stereospecific differences in soluble and membraneous preparations. However, the potency of various catecholamine agonists and the steepness of the displacement curves were greater in all solubilised preparations. Computer-assisted analysis of the displacement curves generated by the highly selective beta₁ antagonist atenolol and the beta₂ antagonist ICI 118.551, revealed the co-presence of beta₁ and beta₂ adrenoceptors in solubilised rabbit lung preparations. Furthermore, soluble beta₁ adrenoceptors appear to be much more labile at 22°C than soluble beta₂ adrenoceptors, providing support for the concept that these receptor subtypes are separate entities.     

Optimal spacer arm microenvironment for the immobilization of recombinant Protein A on heterofunctional amino-epoxy agarose supports

In this study, recombinant Staphylococcus Protein A (rSPA) was immobilized on three different amino-epoxy agaroses: traditional amino-epoxy, butanediol diglycidyl-amino and glycidyl-amino agarose (coded as AE, BDA and GA agarose, respectively), for obtaining affinity adsorbents to bind human immunoglobulin G (hIgG). The effects of the spacer arm microenvironment of the support on the rSPA immobilization were investigated. Compared with the AE agarose, the GA agarose presents ionized amino groups far from the support. Therefore, the rSPA immobilization efficiency of 92 % is slightly higher than that of 88 % on AE agarose due to the weak steric hindrance. Moreover, the BDA agarose exhibited the lowest immobilization efficiency of 58 %, attributing to the existence of hydrophobic butylidene groups on the BDA agarose. Ethanolamine was used as the blocking agent to obtain three affinity adsorbents. The hIgG-binding capacity from the human plasma was determined to be 18.7, 34.7 and 38.7 mg/mL for rSPA-BDA, rSPA-AE and rSPA-GA, respectively. Furthermore, the maximum hIgG-binding capacity was calculated by the Langmuir model of adsorption isotherm to be 25.1, 44.8 and 52.2 mg/mL for rSPA-BDA, rSPA-AE and rSPA-GA, respectively. Therefore, the GA agarose bears the optimal spacer arm microenvironment for preparing the rSPA adsorbent with high hIgG-binding capacity.     

Differentiation of hemagglutinins in tissues infected withChlamydia

Crude, soluble, chlamydial hemagglutinin was prepared from allantoic fluid harvested from embryonated chick eggs and the supernatant fluid of mouse L cells infected with eitherChalamydia psittaci strain 6BC orChlamydia trachomatis strain TW-3. Control nonhemagglutinating specimens of uninfected allantoic fluid and mouse L cells were also prepared. The six preparations were separated by ether-ethanol extraction into lipid-rich and lipid-depleted fractions. Complement-fixing activity was found in the lipid-rich (but not in the lipid-depleted) fraction of infected preparations. In contrast, lipid-rich fractions of infected and uninfected preparations had similar agglutinating activity when sensitive erythrocytes of white Leghorn chickens were used. The lipid-rich fraction of infected and uninfected preparations was separated by thin-layer chromatography (TLC) into seven components with similarR _f values, hemagglutinating patterns, and chemical composition (lipid, protein, and carbohydrate). The highest hemagglutination titers of normal and infected preparations were found in a TLC fraction with similarR _f values and contained lipid, protein, and carbohydrate. This TLC fraction fromC. psittaci 6BC preparations was used in hemagglutination-inhibition studies. The results indicated that chlamydial hemagglutinin extracted by ether-ethanol and separated by TLC contained, in addition to specific hemagglutinin, nonspecific tissue-lipid hemagglutinin(s) identical to that found in normal preparations.     

The influence of sodium dodecyl sulfate from different sources on the separation of virus proteins in polyacrylamide gel electrophoresis

Sodium dodecyl sulfate (SDS) preparations from different sources were investigated with regard to their effect on the separation of the proteins of foot and mouth disease virus (FMDV) types 0₁K, A₂S and C-Obb as well as of Sindbis virus during gel electrophoresis in continuous and discontinuous buffer systems in the presence of 8 m urea. In the continuous system, the different SDS preparations did not markedly alter the separations of any of the virus proteins, but the distances between the FMDV protein bands were small. In the discontinuous system, the source of SDS and the kind of virus protein strongly affected the separations obtained. In this system, adequate resolutions were obtained only with A₂S and C-Obb proteins in the presence of MCB or Pierce SDS preparations. An appreciable percentage of the carbon chains in these SDS preparations were found to be longer than C₁₂. The separations of Sindbis virus proteins which occurred in the continuous buffer PAGE system using any of the SDS preparations were preferable to those which were obtained in the discontinuous system.     

Aged preparations are electrophysiologically more representative of intact tissue than are freshly excised preparations

Studies using excised tissues usually suffer from a discrepancy in the results between freshly excised preparations and aged ones (i.e. preparations which are allowed to rest and-or regenerate for about a day between excision and experimentation). This is especially true for measurements of the membrane potential (V_m) and its changes upon exposure to light in slices of extensor tissue of the laminar pulvini of Phaseolus coccineus L. Measurements of the V _m of extensor cells in situ reveal virtually identifical results to those obtained from aged excised preparations (more negative resting V _m; dramatic, light-induced transients of V _m), whereas freshly excised preparations display a small and light-insensitive V _m. It is concluded, therefore, that for the given example aged preparations are more suitable than fresh ones for investigating the properties of intact tissues.Symbol V_m membrane potential     

Relationship between epitope density and immunoprecipitation of multivalent antigens by bivalent antibody: immunoprecipitation of adenylylated glutamine synthetase by anti-AMP antibodies

Escherichia coli glutamine synthetase (GS) preparations composed of 12 adenylylated subunits (GS₁₂^?) are almost completely precipitated by sheep Anti-AMP immunoglobulin G (IgG), whereas glutamine synthetase preparations containing 6 adenylylated subunits (GS₆^?) are only partially precipitated by the antibodies (R.J. Hohman, S.G. Rhee, and E.R. Stadtman, 1980, Proc. Nat. Acad. Sci. USA77, 7410–7414). By means of ¹²⁵I-labeled anti-AMP antibodies and double immunoprecipitation techniques, in which rabbit antiserum to sheep IgG or anti-GS antibodies were used to precipitate soluble immune complexes, it was demonstrated that under optimal conditions, both the soluble and insoluble immune complexes obtained with either GS₆^? or GS₁₂^? contain 0.5 mol antibody/mol adenylylated subunit. In agreement with the lattice theory of immuno-precipitation, soluble immune complexes are formed in antibody excess. Scatchard plots of binding data indicate that under conditions of antibody excess, one antibody molecule is bound to each AMP moiety of GS₁₂^?, whereas GS₆^? binds a maximum of only 0.68 antibody molecule/adenylylated subunit. We propose that with some species of GS₆^?, the distribution of adenylylated subunits favors monogamous interactions of the bivalent antibody with two subunits within the same GS molecule and thereby leads to the formation of small, soluble, immune complexes. Other explanations are considered. Only 30% of the antibody population that recognizes unconjugated 5′-AMP binds to the AMP moiety of adenylylated GS. Anti-AMP antiserum can be fractionated on a GS₁₂^?-Sepharose matrix into two subpopulations of antibody with strikingly different immunoprecipitation characteristics. Conversely, species of GS with various states of adenylylation ranging from 0 to 8 were separated from a GS₆^? preparation by means of affinity chromatography on an anti-AMP antibody-Sepharose matrix. Under optimal conditions, antibodies purified by affinity chromatography precipitated a smaller fraction of a GS₆^? preparation than did unfractionated antiserum. Competence of the purified antibody was nearly restored to that of the unfractionated serum by the addition of an enhancement factor present in the IgG fraction of nonimmune serum. The enhancement factor was not required for complete precipitation of GS^?₁₂ by purified antibodies. Contrary to most antibody-antigen reactions, immunoprecipitation of GS₆^? with anti-AMP antibodies is greater at 30 °C than at 4 °C.     

Basal serum immunoglobulin levels

Two congenic strains of mice were identified that differ in their serum immunoglobulin levels. The strains were crossed, the F₁ progeny were intercrossed, and the serum immunoglobulin levels of the F₁ and F₂ progeny were analyzed. The F₁ mice have serum immunoglobulin levels like that of the high parent, and the low-immunoglobulin phenotype segregates in the F₂ population. Six other inbred strains of mice were also characterized for basal serum levels of five classes of immunoglobulin.