Direct assay of glycosphingolipid glycosyltransferase activities on thin-layer chromatogram

A modified method for the determination of glycosphingolipid glycosyltransferase activity using high-performance thin-layer chromatographic (HPTLC) plates has been developed. An acceptor glycosphingolipid was chromatographed on an HPTLC plate and was incubated with an enzyme mixture and an appropriate radioactive sugar nucleotide. After incubation, the plate was washed with phosphate buffer and 2% Tween 80. The radiolabeled reaction product was scrapped off the plate and the radioactivity determined using a liquid scintillation counter or, alternatively, the plate was exposed to an X-ray film to reveal the radioactive product. We have used this assay method to determine the activities of rat brain cytidine 5'-monophosphate-N-acetylneuraminic acid: LacCer-, GM3-, GM1-, or GD3-sialyltransferases. This method is sensitive, fast, and reliable and is capable of assaying simultaneously the activities of glycosyltransferases with multiple acceptor specificity. It should be useful in monitoring the enzyme activities present in various column fractions during chromatographic fractionation of glycosyltransferases with different substrate specificities.     

R-ELISA: repeated use of antigen-coated plates for ELISA and its application for testing of antibodies to HIV and other pathogens.

In this paper we report on the evaluation of several procedures that allow for the repeated use of an antigen-coated, enzyme-linked immunosorbent assay (ELISA) plate for enzyme immunoassay (EIA). We have shown that antigen-coated ELISA plates that were incubated once with an aqueous solution containing 8 M urea, 2% sodium dodecyl sulfate and 2% mercaptoethanol, after an EIA, can be reused again for EIA without loss of antigenic capacity. Thus, in this procedure, after an EIA, the ELISA plates were washed once with the above solution and then in a buffer containing 20 mM Tris-HCl, pH 7.5, 0.1% Tween 20 and 500 mM NaCl. This washing protocol was shown to remove the primary antibody, enzyme-conjugated secondary antibody and substrate without removing the antigen from the ELISA plate microwells. Thus, an antigen-coated ELISA plate previously used for an assay could be reused. We tested this repeat ELISA (R-ELISA) procedure on high antigen-binding ELISA plates coated with two different plant virus proteins, a synthetic peptide, the p25/24 gag and the gp120 proteins of the human immuno-deficiency virus, or the staphylococcus enterotoxin protein. In each case tested, the procedure allowed for the repeated use of the same antigen-coated plates for EIA of the respective antibodies. This procedure should prove to be particularly valuable for mass screening of samples tested for HIV and other disease-causing agents.     

A fluorescent plate reader assay for ceramide kinase

Ceramide kinase and its product ceramide 1-phosphate have been implicated in cellular proliferation and survival, activation of cytosolic phospholipase A(2), mast cell degranulation, and phagocytosis. Current assays for ceramide kinase activity employ [(32)P]ATP, with separation of labeled product from excess ATP by organic extraction and thin-layer chromatography. We have developed a fluorescent plate reader assay for ceramide kinase that uses commercially available C6-NBD ceramide (N-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}-D-erythro-sphingosine). Our assay is based on the differential partitioning of substrate and product following a single chloroform/methanol extraction. The product, which partitions into the aqueous phase at physiological pH, is quantitated directly in a plate reader. The substrate may be delivered using either fatty acid-free albumin or detergent/lipid mixed micelles, and we have found that the use of albumin rather than detergent micelles allows one to detect lipid interactions with the enzyme that might otherwise go unnoticed. Our method is useful for assaying ceramide kinase activity both in vitro and in cultured cells, and it offers several advantages over the conventional assay, including greater speed, the ability to run a larger number of assay replicates at one time, and the elimination of environmental and safety issues associated with the use of radioactive materials.     

Use of the enzyme-linked immunoadsorbent assay to monitor the purification of glycosphingolipid antigens by high-performance liquid chromatography

An enzyme-linked immunoadsorbent assay (ELISA) technique has been applied to the analysis of glycosphingolipid fractions separated by high-performance liquid chromatography. Nanogram amounts of selected fractions were placed in microtiter wells and analyzed for glycosphingolipids carrying carbohydrate epitopes recognized by monoclonal antibodies using an avidin-biotin enzyme system (ABC reagents). A large number of fractions (more than 100) can be conveniently evaluated for the presence of glycosphingolipids recognized by one or more monoclonal antibodies in a single analysis. This method is a rapid and sensitive procedure for monitoring the purification of glycosphingolipid antigens and can be used in conjunction with immunostaining of glycosphingolipids separated by thin-layer chromatography.     

2',3'-cyclic nucleotide phosphohydrolase activity determined using an image analyzer detection system

The activity of 2',3'-cyclic nucleotide phosphohydrolase (CNPase) was assayed using high-performance thin-layer chromatography (HPTLC) and an image analyzer detection system. The assay system was used to study a possible inhibitory effect by aminoguanidine on CNPase specific activity. One advantage of using a fixed-time HPTLC system over a real-time spectrophotometric system for an enzyme activity study was that apparent inhibition of the enzyme due to interference of the assay system (chromophore inhibition, etc.) was avoided. In addition, due to the increased accuracy of the image analyzer over conventional methods of TLC plate analysis, a rapid and more accurate measurement of HPTLC plates was possible which required only nanomole amounts of substrate. Also, a digital image of each plate analyzed was stored indefinitely in the computer's memory for future reference. The measurements of CNPase specific activity made using this system compared favorably to those found in recent literature.     

Determination of the number of endothelial cells in culture using an acid phosphatase assay

A simple assay is described in which small numbers of endothelial cells in culture can be determined by measuring acid phosphatase activity. After removal of the growth medium from cells grown in 96-well culture plates, the cells are lysed in buffer containing the detergent Triton X-100 and the phosphatase substrate p-nitrophenyl phosphate. After 2 h at 37 degrees C, the reaction is stopped with sodium hydroxide, and color development is determined using a rapid multiwell plate reader. The assay detects 100 to 10,000 cells per well. The assay has been used to determine growth curves for endothelial cells in the presence and absence of endothelial cell growth factor from bovine hypothalamus and to monitor fractions during purification of the growth factor. Minor modifications in the assay allow it to be fully automated.     

A time-saving thin-layer chromatography plate-scraping system

A system for efficiently scraping thin-layer chromatography plates is described. A height-adjustable rack for holding the thin-layer chromatography plate is constructed of transparent acrylic plate. The base provides a surface on which to slide vial holders and also gives stability to the rack. The plate is scraped with a single-edge razor blade cut to an appropriate width and the scrapings fall into a polypropylene funnel which sits on a vial that is in either a test tube rack or a cassette from a scintillation counter. The plate-scraping system reduces scraping time by more than 60% and increases the accuracy of results.     

Heterogeneity of lipid A: structural determination by 13C and 31P NMR of lipid A fractions from lipopolysaccharide of Escherichia coli 0111

Purified lipid A from Escherichia coli 0111 was fractionated by thin-layer chromatography, and seven major bands were studied by 13C and 31P NMR. All lipid A fractions except one had fatty acids, 3-hydroxytetradecanoic acid, 3-(acyloxy)tetradecanoic acid, and phosphate groups bonded to the diglucosamine backbone. The remaining fraction was shown to be phosphatidylethanolamine. The number of substituents found showed that in all fractions all sites available for C-acylation (C-3, C-4, and C-3') and N-acylation (C-2 and C-2') carried acylic substituents. The number, ranging from four to six, and type of ester-bound carboxylic acid residues as well as the number of phosphate groups differed among the fractions. The three fastest moving bands all had three unsubstituted hydroxy fatty acids and one phosphate group (C-4'), while the slower moving bands had four hydroxy fatty acids and two phosphate groups. Unsubstituted 3-hydroxytetradecanoic acid residues were amide-bound to the disaccharide in all but one of the fractions. In summary, the heterogeneity of E. coli 0111 lipid A is found to be a consequence of a variation of the number and composition of carboxylic acid residues and of varying phosphate content.     

Use of nile red for the rapid in situ quantitation of lipids on thin-layer chromatograms

We describe the use of the fluorescent dye nile red, 9-diethylamino-5H-benzo[alpha]phenoxazine-5-one, as a general-purpose reagent for the rapid detection and quantitation of a wide variety of lipids and other hydrophobic compounds separated by thin-layer chromatography. After samples are applied to silica gel plates and chromatographed, the plate is briefly dipped into a nile red solution (8 micrograms/ml of methanol-water 80:20, v/v). Background fluorescence of nile red dye adsorbed to the silica gel is then preferentially destroyed by dipping the plate in a dilute aqueous solution of bleach. After drying, lipid bands are visualized under ultraviolet light. Reflectance fluorometry (Ex: 580 nm; Em: 640 nm) is utilized for in situ quantitative analysis of the fluorescence of the lipids on the nile red-stained plate. Neutral lipids, phospholipids, sphingolipids, and fatty acids can be examined, although the nile red fluorescence intensity varies significantly among the lipid classes. Also, staining is stronger for unsaturated lipids than for saturated lipids. The lower detection limit of the assay is 25-100 ng for cholesterol, cholesteryl esters, triacylglycerols, and phospholipids.     

Comparative Studies on Plastoquinone II. Analysis for Plastoquinones A, B, C, and D

Two different methods for the extraction and assay of plastoquinones A, B, C and D from chloroplasts of green plants have been described. The long procedure involves separation of aqueous and lipid phases of extract in a separatory funnel, column chromatography, purification on thin-layer plates, and spectrophotometric assays for quantitative determination of the various plastoquinones. The short procedure is based on spotting lipid extracts from chloroplasts on thin layer plates and comparing leucomethylene blue spots of unknown quinones with a series of spots produced by known amounts of the 4 standard plastoquinones on the same plate.

Reliability of the 2 procedures is shown by presenting recovery data (82% recovery for PQ A by the long method and 64-100% recovery by the short method). Various solvent systems for quinone purification are described. Separation of plastoquinones B and C into 6 components each is demonstrated for spinach and a tomato mutant, high pigment (hp). Plastoquinone C is shown to be equivalent to C₁-C₄ while D corresponds to PQ C₅ and C₆ according to Griffiths, Wallwork and Pennock's designation. The term PQ D is therefore redundant and should be abandoned in favor of specific designation of PQ C type.

     

Use of murine myeloma protein M467 for detecting Salmonella spp. in milk

This investigation introduces the use of an immunoglobulin A mouse myeloma protein for the detection of Salmonella spp. in milk. The immunoglobulin A protein M467 reacts with flagellin from a wide variety of serotypes. Two assays were developed which used an enzyme-linked immunosorbent assay (ELISA) and M467. Alkaline phosphatase was conjugated to M467 (M467-PH), and the presence of Salmonella dublin was detected by a competitive solid-phase ELISA and a membrane filtration ELISA. The competitive assay competed viable Salmonella spp. found in contaminated milk against polymerized flagellin or whole bacteria fixed to polyvinyl plates for binding by M467-PH. The membrane filtration method utilized a hydrophilic membrane for filtering the bacteria, which were then detected by the reaction with M467-PH and substrate. The sensitivity of the competitive solid-phase ELISA was 10(3) bacteria ml-1, whereas the filter membrane assay required the media containing the bacteria to be cultured in enrichment medium for 4 h before the assay to ensure detection. Either assay could be run within a typical 8-h work day. The filter membrane assay was not suitable for milk due to the high level of natural alkaline phosphatase activity in the liquid food.     

Use of murine myeloma protein M467 for detecting Salmonella spp. in milk.

This investigation introduces the use of an immunoglobulin A mouse myeloma protein for the detection of Salmonella spp. in milk. The immunoglobulin A protein M467 reacts with flagellin from a wide variety of serotypes. Two assays were developed which used an enzyme-linked immunosorbent assay (ELISA) and M467. Alkaline phosphatase was conjugated to M467 (M467-PH), and the presence of Salmonella dublin was detected by a competitive solid-phase ELISA and a membrane filtration ELISA. The competitive assay competed viable Salmonella spp. found in contaminated milk against polymerized flagellin or whole bacteria fixed to polyvinyl plates for binding by M467-PH. The membrane filtration method utilized a hydrophilic membrane for filtering the bacteria, which were then detected by the reaction with M467-PH and substrate. The sensitivity of the competitive solid-phase ELISA was 10(3) bacteria ml-1, whereas the filter membrane assay required the media containing the bacteria to be cultured in enrichment medium for 4 h before the assay to ensure detection. Either assay could be run within a typical 8-h work day. The filter membrane assay was not suitable for milk due to the high level of natural alkaline phosphatase activity in the liquid food.     

Peroxidase labelled monoclonal antibody against light chains of human cardiac myosin.

Monoclonal antibody against light chains of human cardiac myosin (MLC) was labelled with horseradish peroxidase. The conjugation was performed by two different methods with glutaraldehyde and periodate respectively. The binding activities of the conjugates were tested by enzyme linked immunosorbent assay (ELISA) on the microtitration plates with immobilized MLC (1-1000 ng per well). A comparison of both methods revealed their universal suitability for the preparation of conjugates as well as their applicability. The use of conjugates shortens the time needed and improves the ELISA method for MLC estimation. Specific advantages of the glutaraldehyde and the periodate method concern diverse details.     

A simple and specific assay of glycosyltransferase and glycosidase activities by an enzyme-linked immunosorbent assay method, and its application to assay of galactosyltransferase activity in sera from patients with cancer.

A simple, sensitive, and specific assay method for glycosyltransferase and glycosidase activities has been established by means of an enzyme-linked immunosorbent assay (ELISA) using monoclonal antibody, H-11 directed to lactoneotetraosylceramide (nLc4Cer). Enzyme activity was determined by assaying the amount of reaction product, nLc4Cer with the ELISA method. For the assay of galactosyltransferase activity, lactotriaosylceramide (Lc3Cer) immobilized on a 96-well microtiter plate was incubated with bovine milk galactosyltransferase in cacodylate buffer (pH 6.8) containing Triton CF-54, Mn2+, and UDP-galactose. Optimum incubation conditions for the enzyme were determined. Glycosidase activity was also assayed by the ELISA method by using Clostridium perfringens sialidase and neolacto-series gangliosides as substrates, and the substrate specificities towards the gangliosides were examined. By this method, 3-100 pmol of reaction product could be determined. The assay method has several advantages as follows: 1, the method is simple; 2, separation of the reaction product is not required; 3, quantification and identification of the reaction product were done simultaneously; 4, naturally occurring substrates are available (especially for glycosidase); 5, many samples can be assayed in one microplate; 6, sensitivity is very high. The present method was applied for the detection of galactosyltransferase in human sera. Significant elevations of the galactosyltransferase levels were observed in the sera from cancer patients. The formation of nLc4Cer was confirmed by employing the TLC-immunostaining method for bands of Lc3Cer after incubation of the bands with serum and cofactors on an HPTLC plate.     

Microbial adhesion of Cryptosporidium parvum sporozoites: purification of an inhibitory lipid from bovine mucosa

Cryptosporidium parvum is a protozoan pathogen of humans and livestock worldwide. Its ability to infect a wide range of species raises questions as to the involvement of a specific host cell receptor for parasite-host recognition. To investigate the mechanism of parasite-host cell recognition, we have developed an in vitro cell suspension binding assay to investigate adhesion of C. parvum sporozoites to host cells. Morphologic features of binding events observed with this assay were identical to those described in natural infections. Glycoconjugates, Madin Darby bovine kidney (MDBK) cell fractions, and plasma membrane vesicles (PMVs) were screened for their ability to block binding of sporozoites to MDBK cells. Mucins, MDBK cell fractions, and PMVs exhibited dose-dependent inhibition of sporozoite binding. The major inhibitory fraction from MDBK cells was found to be insoluble in aqueous medium, nonsaponifiable, and lacking carbohydrate moieties, nitrogen, and phosphorus. Its inhibitory effect was resistant to heat, protease digestion, and glycosidase treatment, suggesting that the inhibitory activity is a lipid or a lipid-like component. The inhibitory activity was purified from MDBK cells, and in larger amounts from bovine small intestinal mucosa, by organic solvent extraction, semipreparative high-pressure liquid chromatography, and preparative high-performance thin-layer chromatography. Biochemical analyses, thin-layer chromatography staining techniques, mass spectrometry, and elemental analysis were used to partially characterize the purified lipid. These results indicate that a host intestinal lipid(s) or a lipid-like component(s) may play an important role in the early stages of host cell invasion by C. parvum.     

Biosynthesis of glucosyl monophosphoryl undecaprenol and its role in lipoteichoic acid biosynthesis.

A glucophospholipid was detected in an incubation mixture containing UDP-glucose, MgCl2, ATP, and a particulate enzyme prepared from Streptococcus sanguis. The synthesis of this lipid was inhibited strongly by UDP and moderately by UMP. The molar ratio of glucose to phosphate in the purified lipid was found to be 1:1. Glucose and glucose 1-phosphate were released by mild alkaline hydrolysis of the glucophospholipid. The lipid produced by mild acid degradation of the purified lipid yielded a thin-layer chromatographic profile similar to that of acid-treated undecaprenol. One of the minor components exhibited the same mobility as untreated undecaprenol. To characterize further the lipid moiety of the glucophospholipid, a polyisoprenol was purified from the neutral lipid of S. sanguis. The polyisoprenol was converted in the presence of ATP, UDP-glucose, and the particulate enzyme into a lipid which exhibited the same thin-layer chromatographic mobility as the glucophospholipid. The structure of the polyisoprenol was determined by nuclear magnetic resonance and mass spectrometry to be an undecaprenol with an internal cis-trans ratio of 7:2. These results indicate that the glucophospholipid is glucosyl monophosphoryl undecaprenol. The glucosyl moiety of the glucophospholipid was shown to be incorporated in the presence of the particulate enzyme into a macromolecule which was characterized as a lipoteichoic acid by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and DEAE-cellulose column chromatography. This result indicates that glucosyl monophosphoryl undecaprenol is the direct glucosyl donor in the synthesis of lipoteichoic acid.     

Alkaline butanol extraction of bile salt and steroid sulfate esters: application to the assay of sulfotransferases

A butan-1-ol solvent-extraction procedure has been evaluated for the assay of 3'-phosphoadenosine-5'-phosphosulfate:sulfotransferase activity with various bile salt and steroid substrates. Although butanol extracted the sulfate esters of steroids and bile salts from aqueous solution at neutral pH, extraction at basic pH gave optimum recovery which was independent of protein in the sample. Greater than 99.9% of unreacted 3'-phosphoadenosine-5'-phospho[35S]sulfate remained in the aqueous phase. The data for sulfotransferase activities obtained with this solvent-extraction assay were not significantly different from those obtained with a standard thin-layer chromatography method. Solvent extraction has enabled multiple, rapid assays of several steroid and bile salt sulfotransferases during chromatographic purification of these enzymes from tissue fractions.     

The use of liposomes as acceptors for the assay of lipid glycosyltransferases from rat brain.

Preparation and characterization of sonicated vesicles of various lipid composition containing hydroxy and normal fatty acid ceramides are reported. Such vesicles have been successfully used for the first time as acceptors for the assays of lipid glycosyltransferases, UDP-galactose:ceramide galactosyltransferase and UDPglucose: ceramide glucosyltransferase. Stability of the vesicles and the optimal enzyme activities were the criteria used to select the final composition of the vesicles. The activities of the glycosyltransferases were dependent not only on the appropriate assay conditions but also on the type and source of the phospholipids used to form the liposomes. Ceramides containing normal fatty acids were incorporated into phosphatidylcholine vesicles in a molar ratio of 1 : 3.4 and used as the acceptor for the assay of UDPglucose:ceramide glucostyltransferase. For the assay UDP-galactose:ceramide galactosyltransferase, vesicles were prepared by sonication of bovine brain ethanolamine phospholipids, phosphatidylcholine and ceramide containing alpha-hydroxy fatty acids, in a molar ratio of 6 : 0.57 : 1. The size of the vesicles as determined by electron microscopic measurement ranged mostly between 200--500 A. The results obtained by selective labelling of the outer surface amino groups with the membrane-impermeable reagent, 2,4,6-trinitrobenzenesulfonic acid, indicated that the ethanolamine phospholipid-containing liposomes consisted of closed vesicles. After incubation with the appropriate cofactors and labelled sugar nucleotides, the radioactive reaction products were shown to cochromatograph with the authentic standards by thin-layer chromatography and autoradiography.     

Enzyme-linked immunosorbent assay for human plasma apolipoprotein B

A noncompetitive enzyme-linked immunosorbent assay (ELISA) has been developed for measuring total plasma apolipoprotein (apo) B using affinity purified polyclonal and monoclonal antibodies. Microtiter plates from different manufacturers were tested with regard to their IgG binding characteristics; only one plate yielded consistent coefficients of variation of less than 5%. The optimal plasma dilution in this assay was 1:3000. IgG anti-apoB antisera conjugated to alkaline phosphatase was used as a second antibody. p-Nitrophenyl phosphate was utilized as substrate for color development, and the absorbance (410 nm) was read utilizing an ELISA reader interfaced with a microcomputer for data processing. Plasma apoB levels in plasma have been determined in 1115 male and female participants in the Framingham Offspring Study. Mean (+/- SD) plasma concentrations were 89 +/- 28 mg/dl. Significant age and sex related differences in apoB levels were noted.     

Chemical and biological properties of lipopolysaccharide, lipid A and degraded polysaccharide from Wolinella recta ATCC 33238

Lipopolysaccharide (LPS) was isolated and purified from Wolinella recta ATCC 33238 by the phenol-water procedure and RNAase treatment. The sugar components of the LPS were rhamnose, mannose, glucose, heptose, 2-keto-3-deoxyoctonate (KDO) (3-deoxy-D-manno-octulosonate) and glucosamine. The degraded polysaccharide prepared from LPS by mild acid hydrolysis was fractionated by Sephadex G-50 gel chromatography into three fractions: (1) a high-molecular-mass fraction, eluting just behind the void volume, consisting of a long chain of rhamnose (22 mols per 3 mols of heptose residue) with attached core oligosaccharide; (2) a core oligosaccharide containing heptose, glucose and KDO, substituted with a short side chain of rhamnose; (3) a low-molecular-mass fraction containing KDO and phosphate. The main fatty acids of the lipid A were C12:0, C14:0, 3-OH-C14:0 and 3-OH-C16:0. The biological activities of the LPS were similar to those of Salmonella typhimurium LPS in activation of the clotting enzyme of Limulus amoebocytes, the Schwartzman reaction and mitogenicity for murine lymphocytes, although all the biological activities of lipid A were lower than those of intact LPS.