Detection of cytomegalovirus antibody with latex agglutination.
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Transfusion-acquired cytomegalovirus (CMV) infections should be prevented in seronegative immunocompromised patients by providing blood products from donors who are also seronegative. Latex agglutination was investigated as a simple and rapid method for detecting antibody against CMV. Latex beads were coated with CMV antigen, incubated for 8 min at room temperature with 25 microliter of sera, and examined for agglutination. The sensitivity and specificity of latex agglutination was compared with that of indirect hemagglutination (IHA, Cetus Corp., Emeryville, Calif.) and enzyme immunoassay (EIA) with sera from 604 random blood donors or patients. Of 327 serum samples shown to be seronegative by EIA and IHA, 327 had a latex agglutination titer of less than 1:4 (specificity, 100%). Of 236 serum samples with detectable antibody by EIA and IHA, 228 had a latex agglutination titer of 1:4 or greater (sensitivity, 97%). Plasma collected with EDTA, heparin, or citrate was satisfactory for latex agglutination. Latex agglutination results correlated quantitatively with those of EIA, and the test also detected fourfold or greater rises in antibody with paired sera from six patients with posttransfusion CMV infections. Latex agglutination is a sensitive and specific assay that is rapid and simple to perform and should be effective in selecting seronegative blood donors to prevent posttransfusion CMV infections in seronegative recipients. (+info)
Conditions for haemolysis by flaviviruses and characterization of the haemolysin.
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The 17D vaccine strain of yellow fever virus (YF 17D) was used to establish the optimal conditions for lysis of chick erythrocytes. Tissue culture-grown, polyethylene glycol-concentrated virus showed peak activity at pH 5.4 in citrate buffer when incubated at 37 degrees C. A further two- to fourfold increase in titre was obtained by pretreatment of the chick erythrocytes with 250 micrograms/ml trypsin. These conditions were also shown to be optimal for Japanese encephalitis (JE), West Nile (WN) and dengue-2 (den2) viruses. The ratio of haemagglutination (HA) titre to haemolysis (HL) titre approximated to unity, suggesting that the two functions are associated with the same molecule although as separable entities since selective inactivation of the HL activity of the virus was accomplished using 60 micrograms/ml trypsin. HL could be demonstrated at neutral pH if the chick erythrocytes were first subjected to treatment with acidic pH buffer. The effect on the virus envelope is thus not the sole contribution of a low pH environment to optimal HL. Hyperimmune rabbit antiserum prepared against purified YF 17D virions inhibited HA and HL if added before agglutination had occurred by the virus but when added after agglutination had taken place it showed specific anti-HL activity. Monoclonal antibodies that inhibited HA (HAI) by YF 17D did not inhibit HL (HLI) activity when applied after agglutination had taken place. Moreover, monoclonal antibodies specific for the 54K glycoprotein of YF virus but without HAI activity also had no effect on HL when added either before or after agglutination. As yet, we have been unable to identify a monoclonal antibody displaying specific anti-HL activity but all those directed against the 54K envelope glycoprotein possessing HAI activity showed HA to be a prerequisite for HL. (+info)
Occurrence of O-glycosidically peptide-linked oligosaccharides of poly-N-acetyllactosamine type (erythroglycan II) in the I-antigenically active Sendai virus receptor sialoglycoprotein GP-2.
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Unique high molecular weight (M.W. 4,000-9,000) sugar chains termed erythroglycan II have been obtained from alkali/sodium borohydride digests of I-active asialoglycoprotein derived from sialoglycoprotein GP-2, which was isolated recently from bovine erythrocyte membranes as Sendai virus receptor (Suzuki, Y. et al. (1983) J. Biochem. 93, 1621-1633; (1984) ibid, 95, 1193-1200). It was found that these sugar chains comprise about 40% of total alkali-labile oligosaccharides of asialo GP-2 and contain endo-beta-galactosidase (Flavobacterium keratolyticus)-resistant highly branched and heterogeneous oligosaccharides of poly-N-acetyllactosamine type which are linked O-glycosidically to the peptide backbone through N-acetylgalactosamine. Erythroglycan II also contains endo-beta-galactosidase-susceptible straight terminal polylactosaminyl side chains. A major oligosaccharide released by the enzyme cochromatographed with Gal beta 1-4GlcNAc beta 1-3Gal. Inhibitory activity of Sendai virus-mediated hemagglutination and the receptor activity for the virus were reduced significantly but not completely by the endo-beta-galactosidase. These results indicate that both linear and branched sialosylpolylactosamine sequences in erythroglycan II are important for the reception of the virus into the target cells. (+info)
Coronavirus IBV: removal of spike glycopolypeptide S1 by urea abolishes infectivity and haemagglutination but not attachment to cells.
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Urea has been used to remove the S1 spike glycopolypeptide from avian infectious bronchitis virus (IBV) strains M41 and Beaudette, without removing the S2 spike-anchoring glycopolypeptide. Reduction of the pH to 2.9 did not cause release of S1 although some S1 was released spontaneously from IBV Beaudette at pH 7.4. Virus that lacked S1 was no longer infectious or able to cause haemagglutination (HA). However, radiolabelled IBV that lacked S1 attached to erythrocytes and chick kidney cells to the same or similar extent as did intact virus. Treatment of IBV with a phospholipase C preparation, required to make IBV cause HA, did not increase binding of IBV to erythrocytes. The results indicate that while the attachment to cells of virus that lacks S1 is qualitatively different from that of intact virus, the decline in infectivity is the consequence of the loss of some other spike function. (+info)
Inhibition of BK virus haemagglutination by gangliosides.
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The effect of gangliosides extracted from human group O Rh+ erythrocytes on haemagglutination by BK virus was investigated. Experiments were performed on both ganglioside mixtures and isolated fractions separated by column chromatography and characterized by thin-layer chromatography. These results were compared with those obtained with standard preparations of gangliosides, and the inhibiting activity was shown to be confined mainly to gangliosides with a RF lower than GM1. It was also observed that the insertion of gangliosides in liposomes increased the haemagglutination-inhibiting activity and that ganglioside coating restored the ability of glycosidase-treated human red blood cells to agglutinate. (+info)
Single haemolysis for the assay of antibodies to some haemagglutinating arboviruses.
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The single radial haemolysis reaction has been shown to be suitable for the quantitative assay of specific antibody to West Nile and Sindbis viruses. Only 5 mul of undiluted serum are needed for the test, which can be performed on crude preparations of antigen and without removal of nonspecific inhibitors. It is therefore a very simple procedure. Moreover, it appears to be more specific than the classical haemagglutination-inhibition tests. (+info)
The E3 protein of bovine coronavirus is a receptor-destroying enzyme with acetylesterase activity.
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In addition to members of the Orthomyxoviridae and Paramyxoviridae, several coronaviruses have been shown to possess receptor-destroying activities. Purified bovine coronavirus (BCV) preparations have an esterase activity which inactivates O-acetylsialic acid-containing receptors on erythrocytes. Diisopropyl fluorophosphate (DFP) completely inhibits this receptor-destroying activity of BCV, suggesting that the viral enzyme is a serine esterase. Treatment of purified BCV with [3H]DFP and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the proteins revealed that the E3 protein was specifically phosphorylated. This finding suggests that the esterase/receptor-destroying activity of BCV is associated with the E3 protein. Furthermore, treatment of BCV with DFP dramatically reduced its infectivity in a plaque assay. It is assumed that the esterase activity of BCV is required in an early step of virus replication, possibly during virus entry or uncoating. (+info)
Some factors affecting the production, by cultured baby-hamster kidney cells, of BHK glycoprotein I which cross-reacts immunologically with Tamm-Horsfall glycoprotein.
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Cultured baby-hamster kidney cells (BHK-21/C13), which are adapted to grow in suspension (strain 2P), roduce a glycoprotein, termed BHK glycoprotein I, which cross-reacts immunologically with hamster urinary (Tamm-Horsfall glycoprotein. BHK glycoprotein I was isolated in an electrophoretically (sodium dodecyl sulphate/polyacrylamide gel) homogeneous form by application of affinity chromatography to the medium in which cells had been cultured. Insolubilized anti-(Tamm-Horsfall glycoprotein immunoglobulin G) was used as the adsorbent. The amount of BHK glycoprotein I associated with the cultured cells was found by both radioimmunoassay and immunofluorescence to be related to the amount of Ca2+ in the medium and to the particular stage of the cell cycle. 5'-Nucleotidase was also shed by the cells into the culture medium in amounts related to the stage of the cell cycle. The turnover of hamster Tamm-Horsfall glycoprotein in vivo appeared to be considerably more rapid than can be accounted for by cell turnover. Hamster Tamm-Horsfall glycoprotein was shown to be ineffective in inhibiting agglutination of chicken erythrocytes caused by influenza virus. (+info)