Enzymatic splitting of antibodies bound to immobilized antigen as a means of Fab fragments preparation]

A method of Fab fragments preparation by enzymatic splitting of antibodies bound to specific antigen immobilized on an insoluble support is described. The complex of rat muscle glyceraldehyde-3-phosphate dehydrogenase (GAPD), immobilized on Sepharose 4B, with anti-rat GAPD rabbit antibodies was digested with papain. The antigen was inaccessible to proteolysis under conditions employed. After 4 hrs of incubation with papain the antibody was completely split into non-precipitating fragments. The products of proteolysis not bound to Sepharose, were eluted with 0.1 M givcine buffer pH 2.5, and shown to correspond to Fab fragments.     

Hyaluronate-Binding Proteins of Murine Brain

The distribution of hyaluronate-binding activity was determined in the soluble and membrane fractions derived from adult mouse brain by sonication in low-ionic-strength buffer. Approximately 60% of the total activity was recovered in the soluble fraction and 33% in membrane fractions. In both cases, the hyaluronate-binding activities were found to be of high affinity (KD = 10(-9) M), specific for hyaluronate, and glycoprotein in nature. Most of the hyaluronate-binding activity from the soluble fraction chromatographed in the void volume of Sepharose CL-4B and CL-6B. Approximately 50% of this activity was highly negatively charged, eluting from diethylaminoethyl (DEAE)-cellulose in 0.5 M NaCl, and contained chondroitin sulfate chains. This latter material also reacted with antibodies raised against cartilage link protein and the core protein of cartilage proteoglycan. Thus, the binding and physical characteristics of this hyaluronate-binding activity are consistent with those of a chondroitin sulfate proteoglycan aggregate similar to that found in cartilage. A 500-fold purification of this proteoglycan-like, hyaluronate-binding material was achieved by wheat germ agglutinin affinity chromatography, molecular sieve chromatography on Sepharose CL-6B, and ion exchange chromatography on DEAE-cellulose. Another class of hyaluronate-binding material (25-50% of that recovered) eluted from DEAE with 0.24 M NaCl; this material had the properties of a complex glycoprotein, did not contain chondroitin sulfate, and did not react with the antibodies against cartilage link protein and proteoglycan. Thus, adult mouse brain contains at least three different forms of hyaluronate-binding macromolecules. Two of these have properties similar to the link protein and proteoglycan of cartilage proteoglycan aggregates; the third is distinguishable from these entities.     

Affinity chromatography purification of diphtheria toxin

NAD was covalently linked to Sepharose-4B using a 6 carbon spacer. Sterile, dialyzed spent culture medium containing 100 Lf/ml of diphtheria toxin or material concentrated by (NH₄)₂SO₄ precipitation containing 1500 Lf/ml, was chromatographed on a column of NAD–Sepharose. Ultraviolet absorbing material which did not flocculate with diphtheria antitoxin was eluted with 0.02M phosphate buffer. When the elation buffer was changed to one containing 0.5M NaCl, purified toxin was eluted off the column.     

Evidence for the aggregation of hyaluronateprotein fractioned by chromatography on agarose

Bovine vitreous humour was fractionated by gel filtration chromatography on Sepharose 4B into excluded material, presumably of larger molecular size, and retarded material, presumably of smaller molecular size. No evidence of reversible dissociation of the excluded material on treatment with 300 μM L-ascorbic acid at pH 4.00 or pH 7.40 was detected. The retarded material showed evidence of dissociation when eluted with 1.0 M NaCl, and of association in 0.1 M phosphate buffer pH 7.20. These results are compatible with the hypothesis that hyaluronate molecules are synthesises as smaller units which undergo extracellular association to produce larger molecular aggregates in connective tissues.     

Quantitative Detection of Type A Staphylococcal Enterotoxin by Laurell Electroimmunodiffusion

The detection of staphylococcal enterotoxin A by the quantitative technique of electroimmunodiffusion is described. High dilutions of type-specific rabbit antiserum were used in 1% agarose gels, 1 mm thick, and prepared in 0.05-mug barbital buffer, pH 8.6. Volumes of 10 muliters containing 1.5 to 10 ng of toxin were electrophoresed out of 4-mm diameter wells at 5 mA/cm width of gel. The precipitin cones formed were made visible by first immersing the agarose gels in 0.2 M NaCl and then overlaying the surface with the purified globulin fraction of sheep serum against rabbit globulin, followed by soaking of the gels in 1% aqueous cadmium acetate and staining with 0.1% thiazine red in 1% glacial acetic acid. Fully extended cones, 4 to 23 mm in length depending on toxin concentration and antiserum dilution, were developed in 2 to 5 h of electrophoresis, and visualization was achieved within 2 to 3 h. Because the method is qualitative, quantitative, simple, rapid, and sensitive, it offers a practical tool for the detection of small amounts of bacterial toxins in contaminated foods. The method should also qualify as a sensitive detection device in biochemical procedures which attempt to trace, detect, and identify biological substances in nanogram quantities, provided these substances are antigenic and capable of forming a precipitate with their specific antibodies.     

Conductometric immunosensors for the detection of staphylococcal enterotoxin B based bio-electrocalytic reaction on micro-comb electrodes

Staphylococcal enterotoxin B (SEB) is one of many toxins produced by the Gram-positive bacterium Staphylococcal aureus. While SEB is known as the causative agent of certain food poisonings it is also considered abiological select agent. Thus, rapid and accurate identification of SEB during either surveillance or in response to a biothreat is critical to the mitigation of the suspect agent. This report presents a new conductometric immune-biosensor for the detection of SEB based on immobilization of horseradish peroxidase (HRP)-labeled SEB antibody (HRP-anti-SEB) onto nanogold/chitosan-multiwalled carbon nanotube (Au/CTS-MWNT)-functionalized biorecognition interface. The formation of the antibody-antigen complex by a simple one-step immunoreaction between the immobilized HRP-anti-SEB and SEB in sample solution introduced a barrier of electrical communication between the immobilized HRP and the base surface, thus local conductivity variations could be evaluated by the bio-electrocatalytic reaction of HRP in 0.02 M PBS (pH 6.8) containing 0.15 mM H(2)O(2), 0.06 M KI and 0.1 M NaCl. Under optimal conditions, the proposed immune-biosensor exhibited a good conductometric response relative to SEB concentration in a linear range from 0.5 to 83.5 ng/ml with a correlation coefficient of 0.998. The developed immune-biosensor showed an acceptable accuracy, reproducibility and stability. Milk samples spiked with various concentrations of SEB gave an average of 116% recovery of the toxin.     

Recovery of Campylobacter jejuni and Campylobacter coli from inoculated foods by selective enrichment.

A direct enrichment procedure was developed to selectively recover small numbers of Campylobacter jejuni, C. coli, and nalidixic acid-resistant thermophilic Campylobacter from foods. The procedure includes an enrichment medium composed of brucella broth, 7% lysed horse blood, 0.3% sodium succinate, 0.01% cysteine hydrochloride, vancomycin (15 micrograms/ml), trimethoprim (5 micrograms/ml), polymyxin B (20 IU/ml), and cycloheximide (50 micrograms/ml) that is inoculated with 10 or 25 g of food and incubated with agitation under microaerophilic conditions at 42 degrees C for 16 to 18 h. After incubation, the medium is plated directly onto Campy-BAP agar plates (M. J. Blaser et al., Ann. Intern. Med. 91:179-185, 1979), and resulting colonies that resemble Campylobacter are identified by conventional tests. The foods evaluated included raw milk, hamburger, and chicken skin which had aerobic plate counts of 10(5) to 10(9) bacteria/g. The procedure was effective in recovering as few as 0.1 cell of Campylobacter per g of food. Of the 50 isolates of Campylobacter evaluated, all were recovered from raw milk and hamburger at a level of 1 to 4 cells/g, and 41 and 40 isolaes were recovered from the hamburger and milk, respectively, at 0.1 to 0.4 cell/g. The enrichment was least effective for recovering campylobacters from chicken skin, as 7 and 26 of 50 isolates were not recovered at 1 to 4 and 0.1 to 0.4 cell/g, respectively. This new procedure is more rapid, direct, and effective than other enrichment or direct plating procedures for recovering small numbers of campylobacters from foods.     

Factors Affecting the Secretion of Staphylococcal Enterotoxin A

The biosynthesis of enterotoxin A by replicating and nonreplicating cells was investigated. Unlike enterotoxin B, a secondary metabolite, enterotoxin A secretion resembled that of a primary metabolite by being secreted during the exponential phase of growth. The amount of toxin produced per unit of growth was not influenced by NaCl, NaNO(2), or NaNO(3). Several surfactants increased toxin secretion. Toxin secretion by nonreplicating cells was inhibited by chloramphenicol and 2, 4-dinitrophenol but not by streptomycin or penicillin G. The optimal pH for enterotoxin A production was 6.5 to 7.0. The findings suggest a number of possible reasons for the higher incidence of food poisonings caused by enterotoxin A as compared to enterotoxin B.     

Nonspecific interaction of proteoglycans with chromatography media and surfaces: effect of this interaction on the isolation efficiencies

Nonspecific adsorption of proteoglycans to chromatography media and surfaces is demonstrated. This adsorption is highly dependent on the nature of the chromatography media and the precise buffer conditions. For a given buffer the amount of adsorption decreases as the pH of the buffer is increased. It is also highly dependent on buffer concentration and increases as the buffer concentration is increased. The effect of salts such as LiCl, NaCl, KCl, and MgCl2 was generally small and complex so that the presence of the salt both increased and decreased the amount of adsorption depending on the buffer conditions. In contrast, the effect due to the presence of guanidine hydrochloride (Gdn-HCl) was relatively large and complex. At low Gdn-HCl concentrations there generally was a large increase in the amount of adsorption, reaching a maximum at approximately 0.5 M Gdn-HCl and decreasing with further increases in Gdn-HCl concentration. Detergents such as 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps) and sodium dodecylsulfate generally reduced the amount of nonspecific adsorption, although in the presence of both the detergent and Gdn-HCl, the effect due to Gdn-HCl predominated. In commonly used buffers such as 0.5 M sodium acetate (NaOAc), pH 7.0 (buffer F), and 4 M Gdn-HCl in 0.05 M NaOAc, pH 5.8 (buffer D), adsorption to surfaces and chromatography media such as Sepharose CL-2B, cellulose, and controlled pore glass (CPG) is highly significant and it is particularly large for cellulose and CPG.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of Sodium Chloride and pH on Enterotoxin C Production

Growth and production of enterotoxin C by Staphylococcus aureus strain 137 in 3% + 3% protein hydrolysate powder N-Z Amine NAK broths with 0 to 12% NaCl and an initial pH of 4.00 to 9.83 were studied during an 8-day incubation period at 37 C. Growth was initiated at pH values as low as 4.00 and as high as 9.83 at 0% salt level as long as the inoculum contained at least 10(8) cells per ml. Rate of growth decreased as the NaCl concentration was increased gradually to 12%. Enterotoxin C was produced in broths inoculated with 10(8) cells per ml and above and having initial pH ranges of 4.00 to 9.83, 4.40 to 9.43, 4.50 to 8.55 and respective NaCl concentrations of 0, 4, and 8%. In the presence of 10% NaCl, the pH range supporting enterotoxin C production was 5.45 to 7.30 for an inoculum level of 10(8) cells per ml and 6.38 to 7.30 for 3.6 x 10(6) cells per ml. In repeated experiments in which the inoculum contained 10(8) cells per ml, we failed to demonstrate enterotoxin C production in broths with 12% NaCl and a pH range of 4.50 to 8.55 and concentrated up to 14 times. The effect of NaCl on enterotoxin C production followed the same pattern as its effect on enterotoxin B production. As the concentration of NaCl increased from 0 to 10%, yields of enterotoxin B and C decreased to undetectable amounts.     

Purification of several proteolytic enzymes by tosyl- and carbobenzoxy-triethylene-tetramine-sepharoses.

Tosyl-triethylenetetramine-Sepharose (Tos-T-Sepharose) and carbenzoxytriethylenetetramine-Sepharose (Z-T-Sepharose) were found to be adsorbents utilizable in the purification of several microbial and animal proteases. The former Sepharose derivative adsorbed alpha-chymotrypsin, trypsin, subtilisin, thermolysin and neutral subtilopeptidase at neutral pH range, and acid proteases such as pepsin and Rhizopus niveus protease at pH 3.5-6.5. alpha-Chymotrypsin and trypsin were eluted with 0.1 N acetic acid and Rhizopus protease with 0.5 N acetic acid, thermolysin with 1 M guanidine-HCl or 33% ethyleneglycol, whilst pepsin was recovered by elution with 2 M guanidine-HCl at pH 3.5. The binding of neutral subtilopeptidase and subtilisin to this adsorbent was comparatively weak and both the enzymes were recovered by elution with 0.5 M NaCl at neutral pH. On the other hand, Z-T-Sepharose was found to bind tightly to these proteolytic enzymes except neutral subtilopeptidase. Trypsin and alpha-chymotrypsin were released from the adsorbent column with 1 M p-toluenesulfonate, and subtilisin with 1 M guanidine-HCl or 33% ethyleneglycol at neutral pH region. By these chromatographic procedures, the specific activities of these proteolytic enzymes increased effectively. Comparison of the binding abilities of acetyl-, benzoyl-, tosyl- and carbobenzoxy-T-Sepharoses to these enzymes suggests that hydrophobicity of tosyl and carbobenzoxy groups plays an important role in the enzyme-adsorbent interaction.     

DETECTION OF LISTERIA MONOCYTOGENES BY ADSORPTION AND ELUTION FROM HYDROPHOBIC MATRICES

The binding of L. monocytogenes Scott A strain to three hydrophobic matrices, octyl, phenyl and butyl Sepharose, was investigated. Optimal adsorption of L. monocytogenes to octyl Sepharose was obtained at pH 3.5 and 4 M NaCl. However, it was difficult to elute the bacteria from octyl Sepharose, even after changing the pH and lowering the salt concentration. Good adsorption of L. monocytogenes to phenyl Sepharose at pH 3.5 and 4 M NaCl was also observed. L. monocytogenes was found to adsorb weakly to butyl Sepharose, which is less hydrophobic than phenyl Sepharose. Bacteria were eluted under various conditions. The best elution was obtained with 10 mM sodium phosphate, followed by an increasing gradient of ethylene glycol. To test the potential application of hydrophobic chromatography for separating L. monocytogenes from food matrices, milk was inoculated with L. monocytogenes and then passed through a column of phenyl Sepharose at pH 3.5 and 4 M NaCl. Nearly all L. monocytogenes were bound to the hydrophobic gel and were eluted in a pure and viable form by changing the pH and lowering the salt concentration, and by using a polar reducing agent, ethylene glycol. This study shows that hydrophobic interaction chromatography can be used to separate L. monocytogenes from milk and may be applicable to other food suspensions. It is a gentle method that makes use of the hydrophobic surface properties of Listeria for attachment to hydrophobic gels, as well as using mild elution conditions to avoid inactivation of the organism.     

Adenylate kinase is tightly bound to axonemes of Tetrahymena cilia

Cilia of Tetrahymena thermophila possess adenylate kinase [ATP:AMP phosphotransferase, EC 2.7.4.3] activity. More than 95% of the total activity was recovered in the axonemal fraction when cilia were demembranated with 0.2% Nonidet P-40. There was no loss of the specific activity of adenylate kinase when axonemes were thoroughly washed with HMEK solution (10 mM HEPES, 5 mM MgCl2, 0.1 mM EDTA, and 0.1 M KCl, pH 7.4). These results suggest that adenylate kinase is tightly bound to axoneme. Solubilization of adenylate kinase was markedly increased when axonemes were incubated in HME buffer (10 mM HEPES, 1 mM MgCl2, 0.1 mM EDTA, pH 7.4) containing concentrations of NaCl (or KCl) exceeding 1 M. Therefore, routine isolation of adenylate kinase from axonemes involved pre-extracting axonemes with 0.5 M NaCl in HME buffer followed by extraction in HME buffer containing 1.5 M NaCl. Native-gel electrophoresis of the high salt extract revealed two protein bands (band I and band III). An active staining for adenylate kinase showed a single active band corresponding to the position of band III. Two-dimensional gel electrophoresis using native-gel electrophoresis in the first dimension and SDS-PAGE in the second dimension suggests that band III protein contains at least nine polypeptides ranging from 21 to 110 kDa.     

Partial characterization of thymocyte-activating factor derived from MDP-stimulated guinea pig fibroblasts

The activity of fibroblast-derived thymocyte activating factor (FTAF) of the guinea pig was measured, and the factor was partially characterized. The FTAF activity was heat labile, and destroyed by treatment with trypsin, chymotrypsin, and Streptomyces griseus protease, suggesting the protein nature of FTAF. FTAF bound to DEAE-Sepharose CL-6B in Tris-HCl buffer at pH 8.0, and was eluted with 0.1-0.2 M NaCl. FTAF was absorbed with Blue Sepharose CL-6B. The factor bound to a hydroxylapatite column in 10 mM phosphate buffer and was eluted in two major fractions, one fraction with 40 mM phosphate buffer, the other with 70-110 mM phosphate buffer. Finally, FTAF did not have as much effect on the proliferation of lymph node T cells as T-cell-activating monokines which exhibited marked stimulating effects on both T lymphocytes and thymocytes.     

Simplified purification method for Clostridium botulinum type E toxin.

Clostridium botulinum type E toxin was purified in three chromatography steps. Toxin extracted from cells was concentrated by precipitation and dissolving in a small volume of citrate buffer. When the extract was chromatographed on DEAE-Sephadex without RNase or protamine treatment, the first protein peak had most of the toxin but little nucleic acid. When the toxic pool was applied to a carboxymethyl Sepharose column, toxin was recovered in the first protein peak in its bimolecular complex form. The final chromatography step at 4 degrees C on a DEAE-Sephacel column at a slightly alkaline pH purified the toxin (Mr, 145,000) by separating the nontoxic protein from the complex. At least 1.5 mg of pure toxin was obtained from each liter of culture, and the toxicity was 6 X 10(7) 50% lethal doses per mg of protein. These values are significantly higher than those previously reported.     

Purification, characterization and subcellular localization of pig liver alpha-L-iduronidase

alpha-L-Iduronidase was purified about 100,000-fold from pig liver by employing column chromatography on cellulose phosphate (P11), concanavalin A-Sepharose 4B, heparin-Sepharose 4B, Toyopearl HW-55, Sephadex G-100 and chelating Sepharose 6B charged with cupric ions. The molecular mass of the purified enzyme was estimated to be 70 kDa by Sephadex G-100 column chromatography. The purified enzyme gave a single band on disc polyacrylamide gel electrophoresis without using sodium dodecyl sulfate. However, two separate components of 70 kDa and 62 kDa appeared when it was analyzed by SDS/polyacrylamide gel electrophoresis. These 70-kDa and 62-kDa components were confirmed as alpha-L-iduronidase immunochemically. The isoelectric points of these enzymes were both 9.1 as measured by isoelectric focusing in a polyacrylamide gel containing ampholine and sucrose. The optimal pH and Km values were 3.0-3.5 and 65 microM 4-methylumbelliferyl-alpha-L-iduronide, respectively. The purified enzyme was stable in the pH range 3.5-6.0 under conditions with or without 0.5 M NaCl. However, in the presence of 0.5 M NaCl, it was unstable at pH 3.0. Moreover, it was conversely stabilized at pH 7.0 in the presence of 0.5 M NaCl. Immunohistochemically, the enzyme was found in the Kupffer cells and was abundant on their lysosomal membranes. In liver cells, however, the immunohistochemical reaction was weak.     

Simplified purification method for Clostridium botulinum type E toxin

Clostridium botulinum type E toxin was purified in three chromatography steps. Toxin extracted from cells was concentrated by precipitation and dissolving in a small volume of citrate buffer. When the extract was chromatographed on DEAE-Sephadex without RNase or protamine treatment, the first protein peak had most of the toxin but little nucleic acid. When the toxic pool was applied to a carboxymethyl Sepharose column, toxin was recovered in the first protein peak in its bimolecular complex form. The final chromatography step at 4 degrees C on a DEAE-Sephacel column at a slightly alkaline pH purified the toxin (Mr, 145,000) by separating the nontoxic protein from the complex. At least 1.5 mg of pure toxin was obtained from each liter of culture, and the toxicity was 6 X 10(7) 50% lethal doses per mg of protein. These values are significantly higher than those previously reported.     

Staphylococcal Enterotoxins A and B: Solid-Phase Radioimmunoassay in Food

An immunoassay employing (125)I labeled enterotoxins A and B and polystyrene tubes coated with specific antibodies was used for detection and quantitation of enterotoxin in food. Ham salad, cheddar cheese, custard, condensed milk, and salami were studied. Enterotoxin was successfully determined in all the foods by simple extraction procedures. The assay was sensitive to 1 to 10 ng of toxin per g of food; nonspecific inhibitions were 15% or less.     

Analysis of Staphylococcal Enterotoxin B by the Polyacrylamide Electrophoresis Technique

The electrophoretic mobility of enterotoxin B was investigated through the use of the disc electrophoresis technique. Ideal patterns were developed with a 7.5% acrylamide gel system (pH 4.3). The toxin can be separated and identified from other complex proteins such as serum or suspect samples of foods by this technique. The technique can be used as an assay method for the toxin as well as to elucidate physical changes in the toxin due to temperature. The method should not be considered exclusive for enterotoxin B.     

Affinity chromatography of cysteine-containing histone.

Affinity chromatography based on the reaction between SH groups in protein and +HgC6H4CO groups in the p-mercuribenzoylaminoethyl derivative of Sepharose 4B was examined with a crude preparation of calf thymus cysteine-containing histone. Adsorption of the histone onto the column by specific coupling was found to be optimal in 0.1 M citrate buffer, pH 5.5, containing 5M urea to prevent any aggregation of histones and their non-specific adsorption onto the column, and elution from the column was successfully performed by cleavage of the resulting S-Hg bond with urea-buffer solution containing 0.05 M 2-mercaptoethanol. Under these conditions both the adsorption and elution were quantitative; no adsorption was observed when either SH-blocked histone or unsubstituted Sepharose was used. The cysteine-containing histone thus recovered, after further purification by Bio-Gel P-60 chromatography to remove some cysteine-containing nonhistone proteins contaminating the starting material, showed a single band on polyacrylamide gel electrophoresis and an amino acid composition agreeing with the known sequence of this histone.