Large-Scale Production and Partial Purification of Mouse Immune Interferon

Large-scale production of high-titered (10(2.2) to 10(4) U/ml) immune interferon (type II) was carried out in roller cultures of mouse spleen cells by using the T-cell mitogen staphylococcal enterotoxin A. Precipitation of 90% of this interferon by 55 to 80% saturated ammonium sulfate resulted in a 20-fold concentration and a two- to sixfold purification. After application of this interferon to either bovine serum albumin (BSA)-Affi-Gel 10 or hydroxylapatite columns, 100% of the interferon activity was recovered. By BSA-Affi-Gel 10 chromatography, 7% of the recovered activity was not bound, 45% was eluted with pH gradient 5 to 7, and 48% was eluted with 1 M NaCl. The pH- and salt-eluted interferons from the BSA-Affi-Gel 10 column were purified 62- and 390-fold, respectively, when compared with the starting materials. Rechromatography of the pH- and salt-eluted interferon peaks from the BSA-Affi-Gel 10 column did not alter their elution patterns. Stepwise elution of interferon from the BSA-Affi-Gel 10 columns with buffers of various pH and salt contents also resulted in greater than 300-fold purification. Specific activities of up to 2 x 10(5) U of interferon per mg of protein were attained with either elution procedure from BSA-Affi-Gel 10 columns. By hydroxylapatite chromatography, 5% of the recovered activity was not bound, 20% was eluted with a salt gradient, and 75% was eluted with 30% glycerin. Purification was 107- and 16-fold, respectively, for the two fractions. Ultrogel AcA 34 chromatography of the interferon resulted in two peaks of activity, a major one with a molecular weight of approximately 40,000 and a minor peak of molecular weight 70,000 to 90,000. Thus, by different types of chromatography, immune interferon was found to be heterogeneous.     

Cholesteryl de-esterifying enzyme from Staphylococcus aureus: separation from alpha toxin, purification, and some properties.

A cholesteryl de-esterifying enzyme found in partially purified preparations of alpha toxin produced by the Wood 46 strain on Staphylococcus aureus has been separated from other staphylococcal proteins and from alpha toxin by isoelectric focusing and gel filtration. Preparations of alpha toxin from Bi-Gel P-60 columns and of the cholesteryl esterase from Bio-Gel P-200 columns showed a high degree of purity, as determined by analytical ultracentrifugation, gel diffusion, immunoelectrophoresis, and polyacrylamide gel electrophoresis. The molecular weight of the cholesteryl esterase determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate was 25,500 and on Bio-Gel P-300 columns it was 175,000, indicating an associating system. The density of the enzyme was lower than expected for simple proteins (about 1.19 g/ml). Chloroform-methanol extracts showed the presence of a neutral lipid that did not contain cholesterol. This material, possibly a glycolipid, might play a role in the stabilization of the enzymatically active protomer. The isoelectric point of the esterase was 9.1. Cholesteryl esterase was labile and lost its activity easily. It could bind reversibly to agarose-containing gels. After elution, it was enzymatically inactive, with an isoelectric point of less than 6.2. The W46M mutant of the Wood 46 strain, which does not produce alpha toxin, also does not produce cholesteryl esterase.     

Comparison of some properties of red cell peptidase A from weak and strong activity phenotypes

Partially purified preparations of red-cell peptidase A obtained from individuals with very weak and relatively strong activities have been compared, in order to see whether qualitative differences between the enzymes attributable to different alleles could be identified. It was found that the enzymes obtained both from the weak- and strong-activity phenotypes were very similar in their pH-activity curves, Michaelis constants, and elution profiles from DEAE-cellulose and CM-Sephadex columns. In comparisons of thermostability no marked differences were observed, but there was a suggestion that the enzyme in the weak-activity phenotype may be slightly more thermostable than the enzyme in the strong-activity phenotype.     

Identification and partial purification of pollen allergens from Artemisia princeps

The pollen of Artemisia has been considered as the main late summer-autumn allergen source in this country. To identify its allergenic components, Artemisia princeps pollen extracts were separated by 10% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to nitrocellulose membrane, where IgE binding components were detected by the reaction with sera of twenty Artemisia-allergic patients and 125I-anti-human IgE, sixteen components in the molecular range of 10,000 and 85,000 daltons were detected. Twelve bands bound to IgE from 50% of the sera tested, and two bands (37,000, 23,000 daltons) showed the highest (85%) frequency of IgE-binding in twenty sera tested. When the gel of SDS-PAGE with Artemisia pollen extracts was sliced into 11 allergenic groups (AG) and the protein of each AG was obtained by the gel elution method, the wormwool-RAST inhibition test showed that the AG 10 demonstrated to be the most potent, and the AG 7 was the next. Six AGs showed significant responses (more than 100% of wheal size to histamine, 1 mg/ml) on the skin prick test in more than 50% of the patients tested. It is suggested that electrophoretic transfer analysis with SDS-PAGE may be a valuable method for Artemisia allergen identification, and the possibility of partial purification of allergens by employing gel elution is discussed.     

The effect of storage medium on the elution of monomers from composite materials

The aim of this study was to evaluate the effect of four different storage media on the elution of monomers from two composite materials. Samples (n = 10, diameter: 4.5 mm, thickness: 2 mm) of two different composite materials (Ceram X? & Filtek? Supreme XT) were stored after polymerization in four different media (NaCl, saliva, ethanol 75% & acetone) for 24 h, 7 days, and 28 days. From the storage medium of each tested time period, samples were prepared and analyzed by LC-MS/MS, for the elution of BisGMA, TEGDMA, HEMA, Bisphenol A, and two types of UDMA. No monomers were detected in the samples of Ceram X?, independently of the storage medium used. In the samples of Filtek? Supreme XT, no Bisphenol A, HEMA, and UDMA 1 were found. BisGMA was detected only in the ethanol and acetone samples. The amount of BisGMA eluted in acetone was significant higher compared with ethanol 75% (p < 0.0001). TEGDMA was the only monomer that could be detected in all tested storage media. Storage in acetone resulted in higher release of TEGDMA when compared with other media. The amount of TEGDMA released in saliva was similar to the one released in ethanol 75%. It can be concluded that acetone is not a suitable medium for elution experiments and although ethanol 75% can simulate saliva concerning the elution of TEGDMA, it does not represent a laboratory substitute of saliva with respect to the elution of monomers like BisGMA.     

Fetal-maternal hemorrhage detection in Ontario

The results from fetal-maternal hemorrhage (FMH) detection and quantitation external quality assessment surveys conducted in Ontario indicate that the rosette test had a sensitivity and specificity for an FMH of more than 10 mL of 1.0 and 0.75, respectively, compared with 0.96 and 0.92, respectively, for acid elution. With FMH quantitation, the percentage error of the mean from the target FMH was 20% or more in 7 of 8 surveys, and coefficients of variation ranged from 39.5% to 71.8%. Inadequate Rho(D) immune globulin prophylaxis could have occurred in 19.4% of the challenges with an FMH of more than 10 mL. The rosette and acid elution techniques are both effective for the detection or exclusion of FMH, but acid elution lacks adequate accuracy and precision for reliable FMH quantitation. Furthermore, a strategy of prescribing an extra 1,500-IU Rho(D) immune globulin dose, in addition to the dose required to treat the volume of fetal blood detected, is an effective strategy to overcome the limitations of FMH quantitation by acid elution.     

An improved method for immobilizing IgG antibodies on protein A-agarose

This report describes a modification of a procedure developed by others for crosslinking IgG to protein A which itself is covalently linked to a gel support. Earlier immunoaffinity columns were described as having large antigen-binding capacities and stability under a variety of elution conditions. The present data show that columns constructed with earlier techniques were only partially stable to pH 3.0 buffers, and, as a result, bound less than 20% of the antigen predicted by theory. Modifying parameters of the dimethylpimelimidate crosslinking method led to immunoaffinity columns which did not leak immunoglobulin under low pH elution buffer conditions. The new immunoaffinity absorbants, because of the increased strength of the couple between the antibody and protein A, were capable of binding antigen at over 80% of their theoretical capacity.     

The relative hydrophobicity of oncornaviral structural proteins.

The interaction of the major structural polypeptides from the murine mammary tumor virus (MuMTV) and Rauscher murine leukemia virus (R-MuLV) with alkyl-agarose derivatives containing hydrocarbon arms of various lengths was examined. Both R-MuLV and MuMTV polypeptides were selectively adsorbed to octylimino (C₈)-agarose and decylimino (C₁₀)-agarose columns. Elution of proteins was accomplished using buffers containing 8.5 M ethylene glycol. In certain cases, polypeptides could only be removed from the columns by the addition of detergent to ethylene glycol-containing buffers. By examining the chromatographic behavior of polypeptides using (C₈)- and (C₁₀)-agarose columns and the conditions required for the elution of adsorbed proteins, we were able to determine the relative potential for hydrophobic interaction (degree of hydrophobicity) for each viral protein. The most hydrophobic polypeptide of MuMTV was found to be the glycoprotein gp34, while p15(E) and the gag precursor prp67 appeared to be the most hydrophobic of the R-MuLV proteins. The least hydrophobic polypeptides of MuMTV were p23 and p16, while those of R-MuLV were the two major internal proteins, p10 and p12. Four degrees of apparent hydrophobicity, from least hydrophobic to strongly hydrophobic, could be discerned by this procedure. The major core proteins (MuMTV p28, R-MuLV p30) of both viruses were slightly hydrophobic, while their major glycoproteins were moderately hydrophobic. The degree of hydrophobicity of oncornaviral polypeptides, as determined by hydrophobic chromatography, appeared to bear some relation to the different subviral components with which the proteins were associated. Our results indicate that hydrophobic chromatography may provide a new parameter for the characterization of oncornaviral polypeptides. This procedure should also prove applicable to the study of polypeptides from other viral systems.     

Exploring the feasibility of selective depletion of T lymphocyte subsets by whole blood immunoadsorption cytapheresis

Normal turnover of T lymphocytes is slow relative to other blood cells. Consequently, the physical removal of circulating leucocytes by thoracic duct drainage, repeated leukapheresis or blood filtration results in T cell depletion and immunosuppression. However, clinical use of such procedures is impractical compared with immunosuppressive drugs or radiation. None the less, immunosuppression by physical depletion of T cells, avoiding the systemic toxicities of drugs and radiation, might have clinical advantages if immunophenotypically distinct T cell subsets could be depleted selectively. Recent advances in targeted plasma protein apheresis using adsorbent macrobead columns prompted us to determine whether analogous techniques might permit CD4+ T lymphocytes to be removed selectively from whole blood. To explore this possibility, we linked murine anti-human-CD4 and isotype-identical control monoclonal antibodies (mAbs) to agarose, polyacrylamide and polystyrene macrobeads (150-350 microm) and then evaluated the selectivity, specificity and efficiency of macrobead columns to remove CD4+ T cells from anti-coagulated whole blood at varying mAb densities and flow rates. We also examined saturation kinetics and Fc-oriention versus random coupling of mAbs to macrobeads. Sepharose 6MB macrobead (250-350 microm) columns proved to be most effective, selectively removing up to 98% of CD4+ T cells from whole blood. Moreover, depletion efficiency and selectivity were retained when these columns were reused after elution of adherent CD4+ cells. These studies indicate that selective depletion of T lymphocyte subsets by whole blood immunoadsorption apheresis using mAb-linked macrobead columns may be feasible on a clinical scale. It is possible that such apheresis techniques could achieve targeted forms of immunosuppression not possible with drugs or radiation.     

Purification of horse immunoglobulin isotypes based on differential elution properties of isotypes from protein A and protein G columns

Elution properties of horse immunoglobulin isotypes from protein A and protein G columns were examined. IgGa and IgGb isotypes were bound to protein A and protein G columns and were eluted by adjusting the pH of the elution buffer from 8.0 to 2.0. IgGc bound to protein G column but not to protein A column while IgG(T) bound to both columns. IgM and IgA apparently appeared not to bind to either column. New methods for purification of serum isotypes were developed using protein A and protein G columns as well as formerly established methods. Using these methods, it was possible to obtain purified isotypes for establishment of immunological assays for practical clinical use.     

Immunochemical estimations of allergenic activities from outdoor aero-allergens, collected by a high-volume air sampler

To quantify airborne allergens in amorphus and morphological particles, a survey with collection of aero-allergens on glass fibre filters by means of a high-volume air-sampler (HIVOL) was conducted. In preliminary laboratory experiments we compared various filter elution techniques, and the pulverizing elution technique was found to be optimal with regard to yield and convenience. When a surfactant, Tween 20 (0.5% v/v), was added to the elution buffer, a recovery of 80% could be obtained. Allergens in eluates were analysed by means of an IgG-subclass RAST inhibition assay. This immunochemical method for quantification of airborne allergens was validated, as a high recovery of timothy grass pollen allergens was eluted from air filters, and eluates were shown specific by RAST inhibition. The amount of immunochemically measured airborne timothy and birch allergens collected by means of the HIVOL sampler was highly correlated with pollen counts obtained with a Burkard sampler (pollen trap) situated in the same place.     

Purification and characterization of mouse beta-2 microglobulin: allelic variants from two different strains

Beta-2 microglobulin (beta 2M) is a 12,000 dalton protein associated with membrane-bound cell surface antigens. Variants of beta 2M, beta 2MA and beta 2MB, were first detected by Michaelson et al. (Immunogenetics 11, 93-95, 1980). An improved method was used to purify beta 2MA and beta 2MB from BALB/c and C57BL/6 mouse livers, respectively. Reproducible yields of 10% were obtained. The purifications were accomplished by a 3 M sodium thiocyanate (NaSCN) extraction of a crude membrane fraction, an acid precipitation step, gel filtration on Sephadex G-75 and ion-exchange chromatography on DEAE-cellulose and CM-cellulose in that order. The elution profile of beta 2MA and beta 2MB on the ion-exchange columns was found to be different, indicating the presence of structural changes. beta 2MA was found to be more acidic (pI = 7.35) than beta 2MB (pI = 7.68) by isoelectric focusing in gels. Complete sequence analysis of beta 2MA and partial sequence analysis of beta 2MB (61 of 99 residues) were performed by automated Edman degradation of the intact chain and of the overlapping peptides obtained by: (a) tryptic cleavage at arginines after acetimidation of lysine side chains, (b) BNPS-skatole cleavage at tryptophan residues and (c) hydroxylamine cleavage at asparagine-glycine linkages. A comparison of the primary structure of beta 2MA to the partial amino acid sequence obtained for beta 2MB revealed a single amino acid substitution (aspartic acid for alanine at position 85) that accounts for the differences in biochemical properties observed.     

The effect of sodium thiocyanate on virus structure.

A study of two different virus types after elution from immunosorbent columns by sodium thiocyanate has shown that virus degradation occurs. The two virus types studied were hepatitis B and rotavirus. Hepatitis B antigen (HBAg) was only slowly degraded and retained many of its morphological features, although in an altered form; rotavirus was highly sensitive to the chaotropic agent, losing both its viability and its morphological integrity. During the process of disassembly a previously undetected inner component of rotavirus could be visualised, which was termed the "core."     

Viral elution and concentration method for detection of influenza A viruses in mud by real-time RT-PCR

The role of environmental reservoirs in avian influenza virus (AIV) transmission has been investigated during AIV-associated outbreaks. To date, no method has been defined for detection of AIV from mud samples. A procedure using elution and polyethylene glycol (PEG) concentration steps was designed to detect AIV by RT-PCR from 42 g of raw mud, corresponding to 30 g of the solid fraction of mud. RNA was recovered with MagMAX AI/ND Viral RNA Isolation kit (Ambion, Austin, TX). Three elution buffers were studied and viral recoveries higher than 29% were yielded by elution with a 10% beef extract solution (pH 7). The overall method showed that, under some conditions, virus was not detectable in PEG samples, whereas viruses were detected in the elution fractions. PCR curves were improved significantly by running the amplification reaction with a mixture containing a PCR additive for inhibitor removal, such as T4 gene 32 protein (Gp32), although PCR inhibitors from mud were removed partially from PEG samples. A theoretical detection threshold of 5 × 10⁵ RNA copies of H5N1 virus per 30 g of solid mud could be obtained by elution. The overall method has proved successful for detecting H5N1 virus contamination of mud specimens collected during outbreak investigations of avian influenza in Cambodia.     

Isolation of antibody to human placental alkaline phosphatase (PLAP) from extracts of human placentae

PROBLEM: Human placental alkaline phosphatase (PLAP) is a unique placental antigen bound to the syncytiotrophoblast, which may be able to elicit a specific immune response in pregnancy. METHOD OF STUDY: Antibody to PLAP was purified from placental extracts by: a) acid elution of membrane vesicles; b) purifying complexes of PLAP with human antibody on monoclonal antibodies to PLAP followed by denaturation of the enzyme; and c) by denaturation of PLAP in placental extracts and purification of antibody to PLAP on PLAP columns. RESULTS AND CONCLUSIONS: Specific antibody to PLAP is present in placental extracts, and is mostly bound to placental membrane preparations. PLAP is therefore immunogenic in pregnancy and could serve as a useful monitor of pregnancy-specific immunological responses. Since a similar enzyme appears in some cancers, it is possible that the immunization against PLAP in pregnancy will help to protect against the development of ovarian and endometrial cancer (the 'fetal antigen' hypothesis).     

Associated expression of CD1 antigen and Fc receptor for IgE on epidermal Langerhans cells from patients with atopic dermatitis.

The presence of Fc receptors for IgE on epidermal Langerhans cells (LC) from patients with atopic dermatitis (AD) was demonstrated by three different types of experiments. Firstly, cell-bound IgE on LC was removed by acid elution and restored by highly purified human myeloma IgE (IgE kappa). Secondly, after pepsin digestion of cell-bound IgE the number of LC staining with anti-human light chain (kappa, lambda) antibodies significantly decreased in contrast to the number of LC staining with anti-human epsilon heavy chain antibody. Thirdly, LC formed rosettes with sheep red blood cells (SRBC) coated with IgE kappa. Epidermal LC from normal non-atopic controls, did not form rosettes with SRBC-IgE. The SRBC-IgE rosette formation could be inhibited by preincubation with IgE kappa and BB10 (MoAb directed against the Fc receptor for IgE on human eosinophils, platelets and macrophages), but also with human IgG, whereas the SRBC-IgG rosette formation could be inhibited neither by IgE kappa nor by BB10. Both the SRBC-IgE and the SRBC-IgG rosette formation could be inhibited by OKT6 (anti-CD1) antibody. The results of inhibition studies with OKT6 antibody on the reconstitution of IgE on epidermal LC after acid elution suggest an associated expression of the CD1 antigen and the Fc receptor for IgE.     

In Vitro Generation of Eosinophil Chemotactic Factor from Human and Murine Mononuclear Phagocytes

Low molecular weight eosinophil chemotactic factor (ECF), which has previously been demonstrated in mast cells, basophils, neutrophils and eosinophils, was shown to be released by several types of mononuclear phagocytes. Highly purified rat peritoneal macrophages and human monocytes produced ECF on stimulation with the calcium ionophore A23187 (Ion) and with phagocytic stimuli in a time-dependent fashion, whereas lymphocyte- or mast cell-specific stimuli were ineffective. Two murine macrophage lines and a fibroblast cell line (L cells) also generated and secreted ECF with the different stimuli. ECF from macrophages was similar to that from neutrophils in its target cell specificity (eosinophils and neutrophils) and its elution profile on Sephadex G-10 columns (300-500 dalton). ECF secretion from monocytes was not affected by mitomycin C or cycloheximide, whereas indomethacin enhanced and a phospholipase A inhibitor decreased its production. These in vitro findings suggest that, through ECF, mononuclear phagocytes may potentially regulate eosinophil and neutrophil influx to sites of inflammatory reactions.     

Bacteriophage concentration from water by filter chromatography

The efficiency of an electropositive filter for membrane chromatography of viruses was examined using coliform phages T1, T4, lambda and Salmonella phage P22. Phages diluted in dechlorinated tap water were adsorbed to filters at neutral pH and eluted by 3% beef extract in 0.05 M glycine buffer at selected alkaline pH values. With exception of lambda phage, which displayed erratic adsorption behavior at any pH, all bacteriophages studied, adsorbed to filters with an efficiency of 97-100% at pH values ranging between 6.0 and 8.0. Each phage was readily eluted at alkaline pH levels. Maximal elution (86.2%) of T1 phage and lambda phage (79%) occurred at pH 10, while T4 and Salmonella phages were eluted most efficiently at pH 11 at values of 91.7 and 81.9%, respectively. The resolving power of the filter was such that individual phages within the same virus group (T1 and T4 phage) could be eluted at pHs differing by only one unit.     

Quantitation of fetal-maternal hemorrhage by flow cytometry. A simple and accurate method

A simple and objective assay was developed for the detection and quantitation of fetal-maternal hemorrhage with the use of flow cytometry. In vitro prepared control mixtures of 10%, 2%, 1%, 0.5%, 0.25%, 0.125%, and 0.06% D+ RBCs in D- RBCs were tested (8-11) different times by flow cytometry and gave mean % D+ results of 11.10%, 1.90%, 0.92%, 0.45%, 0.24%, 0.11%, and 0.05%. The coefficient of variation of preparing and testing these mixtures ranged from 11.0 to 15.9% for the 10-0.125% mixtures. Thus, flow cytometry was accurate, reproducible, and sensitive. Flow cytometry was compared with Du tests, rosette tests, and acid elution. The Du test was highly variable because it was not sensitive enough to detect a significant bleed (approximately 0.6%) in some cases and too sensitive (necessitating quantitation of an insignificant bleed) in others. The rosette test was too sensitive. Acid elution and flow cytometry results did not always agree; acid elution results were approximately twice as high as flow cytometry. The authors believe flow cytometric detection of D+ red blood cells to be more accurate than the detection of fetal hemoglobin by acid elution techniques, which is known to have poor reproducibility. Postpartum samples from 56 D- women who delivered D+ babies were tested. Fifty-two had fetal bleeds less than 0.3% by acid elution and flow cytometry; all had negative Du test results, but there were two false positive results with the use of the rosette technique. Four had significant bleeds (greater than or equal to 0.6%); in all four cases the flow cytometry results were lower than the acid elution results. The authors were able to quantitate a bleed of fetal RBCs, which were D+ only by the Du test, in a D- mother with the use of flow cytometry, and D+ RBCs in a mother whose RBCs were of the rare DVI mosaic phenotype. This would not have been possible with the use of the standard Du or rosette techniques.     

Use of molecularly imprinted polymers in the solid-phase extraction of clenbuterol from animal feeds and biological matrices

Clenbuterol molecularly imprinted polymers (MIPs) as chromatographic stationary phase for the solid-phase extraction (SPE) of the drug from biological samples have been prepared. Propylene columns filled with 500 mg of clenbuterol MIPs have been tested with respect to their loading capacity, memory effects, selectivity toward related drugs (mabuterol, clenproperol, clenisopenterol, ritodrine) and specificity toward interferences arising from heterogeneous matrices such as animal feeds, bovine urine and liver. Analytes were concentrated on Extrelut 20 columns and the residues resuspended in 70% acetonitrile. Application, washing and elution fractions were collected and analyzed by HPLC-diode array detection. Results indicate this MIP approach in SPE is extremely selective for clenbuterol, mabuterol, clenproperol and clenisopenterol (>95% found in the eluate), with a loading capacity of about 20 microg/100 mg of stationary phase. Ritodrine showed a recovery rate of 51%. The molecular recognition mechanism is so specific to allow clenbuterol detection and identification by conventional detectors at level of interest (ppb) also from complex matrices such as feeds, urine and liver.