Showing papers on "Hemagglutination published in 2009"

Mechanisms of the action of povidone-iodine against human and avian influenza A viruses: its effects on hemagglutination and sialidase activities

Abstract: Influenza virus infection causes significant morbidity and mortality and has marked social and economic impacts throughout the world. The influenza surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA), act cooperatively to support efficient influenza A virus replication and provide the most important targets for anti-influenza chemotherapy. In this study, povidone-iodine (PVP-I), which has a broad-spectrum microbicidal property, was examined for its inhibitory effects against influenza virus infection in MDCK cells and the mechanisms of PVP-I action on HA and NA were revealed. Results obtained using a novel fluorescence- and chromogenic-based plaque inhibition assay showed that 1.56 mg/ml PVP-I inhibited infections in MDCK cells of human (8 strains) and avian (5 strains) influenza A viruses, including H1N1, H3N2, H5N3 and H9N2, from 23.0–97.5%. A sialidase inhibition assay revealed that PVP-I inhibited N1, N2 and N3 neuraminidases with IC50 values of 9.5–212.1 μg/ml by a mixed-type inhibition mechanism. Receptor binding inhibition and hemagglutinin inhibition assays indicated that PVP-I affected viral hemagglutinin rather than host-specific sialic acid receptors. Mechanisms of reduction of viral growth in MDCK cells by PVP-I involve blockade of viral attachment to cellular receptors and inhibition of viral release and spread from infected cells. Therefore, PVP-I is useful to prevent infection and limit spread of human and avian influenza viruses.

Sensitized sheep red cells as a reactant for Staphylococcus aureus protein A. Methodology and epidemiology with special reference to weakly reacting methicillin-resistant strains.

Abstract: Staphylococci with cell-bound protein A reacted with sensitized sheep red cells in a simple slide reaction with agglutination. Extracellular protein A proved capable of preventing the sensitized sheep red cells from reacting with cultures of Staph, aureus strains containing protein A. This confirms the reactivity of protein A with free Fc-fragments of IgG on the surface of the sensitized sheep red cells as the cause of haemagglutination. The choice of culture medium proved important for demonstrating protein A. Peptone-free media proved clearly unsuitable. 341 routinely isolated strains of Staph. aureus were examined for agglutination of sensitized sheep red cells in the slide test and for tube-agglutination of sensitized sheep red cells with broth culture filtrate. 88.3 per cent of the strains were positive if examined on slides, while 6.7 per cent of the strains contained no cell-bound or free protein A demonstrable by either method. Among the methicillin resistant strains, 50 per cent showed no agglutination on slide. Antibiotic-sensitive strains belonging to phage group II tended to have weaker protein A reactivity than other groups.

An Evaluation of Avian Influenza Diagnostic Methods with Domestic Duck Specimens

Abstract: SUMMARY. Monitoring of poultry, including domestic ducks, for avian influenza (AI) virus has increased considerably in recent years. However, the current methods validated for the diagnosis and detection of AI virus infection in chickens and turkeys have not been evaluated for performance with samples collected from domestic ducks. In order to ensure that methods for the detection of AI virus or AI virus antibody will perform acceptably well with these specimens, samples collected from domestic ducks experimentally infected with a U.S. origin low pathogenicity AI virus, A/Avian/NY/31588-3/00 (H5N2), were evaluated. Oropharyngeal (OP) and cloacal swabs were collected at 1, 2, 3, 4, 5, 7, 10, 14, and 21 days postinoculation (PI) for virus detection by virus isolation, which was considered the reference method, and real-time RT-PCR. In addition, two commercial antigen immunoassays were used to test swab material collected 2–7 days PI. Virus isolation and real-time RT-PCR performed similarly; however, the antigen immunoassays only detected virus during the peak of shed, 2–4 days PI, and both kits detected virus in fewer than half of the samples that were positive by virus isolation. Cloacal swabs yielded more positives than OP swabs with all virus detection tests. To evaluate AI virus antibody detection serum was collected from the ducks at 7, 14, and 21 days PI and was tested by agar gel immunodiffusion (AGID) assay, a commercial blocking enzyme-linked immunosorbent assay (ELISA), and homologous hemagglutination inhibition (HI) assay, which was used as the reference method. Results for the ELISA and HI assay were almost identical with serum collected at 7 and 14 days PI; however, by 21 days PI 100% of the samples were positive by HI assay and only 65% were positive by ELISA. At all time points AGID detected antibody in substantially fewer samples than either ELISA or HI assay.

Biochemical characterization of the feline AB blood group system.

Abstract: Summary The biochemical nature of the feline AB blood group system was characterized by analysing red blood cells from homozygous (genotype A/A) and heterozygous (A/B) type A, type B (B/B), and type AB cats. High performance thin layer chromatography (HPTLC) of red cell gly-colipids revealed that specific neuraminic acids (NA) on gangliosides, containing ceramide dihexoside (CDH) as a backbone, correlated with the feline AB blood group antigens. Although disialogangliosides predominated, mono- and trisialogangliosides were also isolated. B cats expressed solely N-acetyl-NA (NeuNAc) on these gangliosides. In addition to expressing N-glycolyl-NA (NeuNGc) containing gangliosides, A red cells have gangliosides with only NeuNAc or mixtures of both NA. HPTLC profiles of disialogangliosides from homozygous and heterozygous A cats differed slightly in the quantity of disialogangliosides. Equal amounts of NeuNAc and NeuNGc containing disialogangliosides, as well as two intermediary forms, were recovered from AB erythrocytes. Analysing disialogangliosides from red cells belonging to 17 genetically related cats, we consistently obtained the expected disialoganglio-side profile, based on blood typing and pedigree information. SDS-PAGE of red cell membrane proteins and blotting with Triticum vulgaris, a lectin recognizing NeuNAc, revealed glycoproteins of approximately 51, 53, and 80 kD in B and AB cats but only a faint band of approximately 53 kD in A cats. By haemagglutination, Triticum vulgaris could also distinguish different blood types by specifically binding to B and AB cells. Flow cytometry showed that more anti-B bound to B cells than anti-A bound to A cells. Although AB cells had a broad range of fluorescence when compared to the profiles seen with A and B cells, the mean fluorescence with AB cells was half of that seen with A or B. These results further characterize the antigens determining the feline AB blood group system illustrating differences between A, B and AB cats, and between homozygous and heterozygous A cats.

Transfusion in the age of molecular diagnostics

Abstract: DNA-based tests are increasingly being used to predict a blood group phenotype to improve transfusion medicine. This is possible because genes encoding 29 of the 30 blood group systems have been cloned and sequenced, and the molecular bases associated with most antigens have been determined. RBCs carrying a particular antigen, if introduced into the circulation of an individual who lacks that antigen (through transfusion or pregnancy), can elicit an immune response. It is the antibody from such an immune response that causes problems in clinical practice and the reason why antigen-negative blood is required for safe transfusion. The classical method of testing for blood group antigens and antibodies is hemagglutination; however, it has certain limitations, some of which can be overcome by testing DNA. Such testing allows conservation of antibodies for confirmation by hemagglutination of predicted antigen-negativity. High-throughput platforms provide a means to test relatively large numbers of donors, thereby opening the door to change the way antigen-negative blood is provided to patients and to prevent immunization. This review summarizes how molecular approaches, in conjunction with conventional hemagglutination, can be applied in transfusion medicine.

Application of the passive haemolysis test for the determination of rubella virus antibodies.

Abstract: A passive haemolysis test for the determination of antibodies against rubella virus haemagglutinin is presented. According to this method, the principle of radial immunodiffusion technique is applied. Rubella haemagglutinin-coated chick erythrocytes in the agarose gel were lysed by the diffusing positive sera in the presence of complement at 37° C. The passive haemolysis test was compared with the conventional haemagglutination inhibition method, and the diameter of the haemolysis zones was shown to be a direct measure of the quantity of antibody added to the well. A plot of the log (HI titre) against the zone diameter gives a straight line. There is no need to remove nonspecific haemagglutination inhibitors. However, all serum samples must be inactivated at 56° C for 30 minutes before testing. Tests of 200 serum samples from healthy women showed a good correlation between the haemagglutination inhibition titre and the antibody titre determined by the passive haemolysis technique. Twenty-one samples with a haemagglutination inhibition titre less than 10 were also negative in the passive haemolysis test. With the exception of one, all sera with a positive haemagglutination inhibition titre showed a positive haemolysis reaction. The method was found to be as specific and as sensitive as the haemagglutination inhibition test. In addition, the technique is rapid and simple for quantitative studies of antibodies against rubella virus.

Detection of human antibodies to Trichinella spiralis by enzyme-linked immunosorbent assay, ELISA.

Abstract: Enzyme-linked immunosorbent assay, ELISA, was used to detect and quantitate antibodies to Trichinella spiralis in human sera. An extract of Trichinella spiralis larvae was adsorbed to polystyrene tubes. These were incubated with serum and bound antibodies were detected by anti-immunoglobulin labelled with alkaline phosphatase. Class specific conjugates were used to detect specific IgM and IgA antibodies. ELISA was at least as sensitive as passive haem-agglutination and more sensitive than indirect immunofluorescence.

Development of a real-time polymerase chain reaction assay for the simultaneous detection of Mycoplasma gallisepticum and Mycoplasma synoviae under industry conditions

Abstract: In this research we developed a real-time SYBR green assay to detect both Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) in a single reaction. A total of 30,000 samples from broiler breeder flocks were screened using traditional serology (plate agglutination, enzyme-linked immunosorbent assay, hemagglutination inhibition) and polymerase chain reaction (PCR; traditional and real-time). It was determined that the real-time SYBR green PCR assay developed in this research was more rapid than all three methods tested and more sensitive and specific than culturing or serology. The SYBR green assay was optimized and could detect as few as 30 template copies of DNA per sample. In addition, the SYBR green assay was less expensive than traditional culturing and serology. MG and MS are infectious bacteria that can rapidly spread and infect commercial chicken flocks. These diseases can cause a significant loss to the poultry industry and especially to broiler breeders because infected flocks are destroyed under the National Poultry Improvement Plan MG and MS clean programs. The real-time SYBR green assay developed in this research has the potential to reduce the time it takes to reach a correct diagnosis and to arrest outbreaks of MG and MS.

Synthesis of sialoglycopolypeptide for potentially blocking influenza virus infection using a rat α2,6-sialyltransferase expressed in BmNPV bacmid-injected silkworm larvae

Abstract: Sialic acid is a deoxy uronic acid with a skeleton of nine carbons which is mostly found on cell surface in animals. This sialic acid on cell surface performs various biological functions by acting as a receptor for microorganisms, viruses, toxins, and hormones; by masking receptors; and by regulating the immune system. In order to synthesize an artificial sialoglycoprotein, we developed a large-scale production of rat α2,6-sialyltransferase (ST6Gal1). The ST6Gal1 was expressed in fifth instar silkworm larval hemolymph using recombinant both cysteine protease- and chitinase-deficient Bombyx mori nucleopolyhedrovirus (BmNPV-CP--Chi-) bacmid. The expressed ST6Gal1 was purified, characterized and used for sialylation of asialoglycopolypeptide. We tested the inhibitory effect of the synthesized α2,6-sialoglycopolypeptide on hemagglutination by Sambucus nigra (SNA) lectin. FLAG-tagged recombinant ST6Gal1 was expressed efficiently and purified by precipitation with ammonium sulphate followed by affinity chromatography on an anti-FLAG M2 column, generating 2.2 mg purified fusion protein from only 11 silkworm larvae, with a recovery yield of 64%. The purified ST6Gal1 was characterized and its N-glycan patterns were found to be approximately paucimannosidic type by HPLC mapping method. Fluorescently-labelled N-acetyllactosamine (LacNAc) glycoside containing dansyl group was synthesized chemo-enzymatically as high-sensitivity acceptor substrate for ST6Gal1. The acceptor substrate specificity of the enzyme was similar to that of rat liver ST6Gal1. The fluorescent glycoside is useful as a substrate for a highly sensitive picomole assay of ST6Gal1. Asialoglycopolypeptide was regioselectively and quantitatively sialylated by catalytic reaction at the terminal Gal residue to obtain α2,6-sialoglycopolypeptide using ST6Gal1. The α2,6-sialoglycopolypeptide selectively inhibited hemagglutination induced by Sambucus nigra (SNA) lectin, showing about 780-fold higher affinity than the control fetuin. Asialoglycopolypeptide and γ-polyglutamic acid did not affect SNA lectin-mediated hemagglutination. The recombinant ST6Gal1 from a silkworm expression system is useful for the sialylation of asialoglycopeptide. The sialylated glycoprotein is a valuable tool for investigating the molecular mechanisms of biological and physiological events, such as cell-cell recognition and viral entry during infection.

Cross-reactivity of bacteroides fragilis o antigens

Abstract: The cross-reactivity of O antigens prepared from Bacteroides fragilis, other Bacteroides species and from Fusobacterium has been examined by indirect haemagglutination and inhibition of haemagglutination. Fifteen of 20 B. fragilis ss. fragilis strains showed O-antigenic cross-reactivity with one or more of the test strains of B. fragilis ss. fragilis: NCTC 9343, Lille E 323 and SBL B55. The same applies also to 3 strains classified as B. coagulans, B. hypermegas and B. putredinis. The multispecificity of B. fragilis O antigens is pronounced. Test systems for demonstration of 9 specificities, all harboured by one or more of the 3 test strains, have been worked out.

Comparative efficacy of peste des petits ruminants (ppr) vaccines available in pakistan in sheep and goats

Abstract: The efficiency of Peste des petits ruminants virus (PPRV) vaccines available in Pakistan was evaluated on the basis of the humoral immune response measured by haemagglutination inhibition (HI) and agar gel immunodiffusion (AGID) tests in sheep (n=60) and goats (n=60). The effect of storage temperature on HA activity of vaccine virus was measured by holding the vaccine at -20, 4, 27 and 40°C for 24 hours. The titer of freshly prepared vaccine was 1:16 and remained unchanged for 24 hours in the vaccines stored at 20 and 4°C. However, drop in titer (1:2 HA) was recorded in the vaccine kept at 40°C for 24 hours. The haemagglutination activity of PPR virus constituted in buffer with pH 6.8 and 7.0 was recorded as highest when assay was performed with chicken and human blood group’O’ erythrocytes (1%). The lowest titer was recorded when vaccine was reconstituted in buffer at pH 8.0. After 14 th day post vaccination, there was a gradual increase in the antibody titer till 56 th day. Geometric mean titer (GMT) of antibodies against locally manufactured PPRV vaccine was higher (207.9) in comparison with Pestivec (73.3), a vaccine imported from Jordan at 63 rd day post vaccination in sheep; the corresponding values in goats were 147.0 and 48.5, respectively. All animals of control group were negative for antibodies by both of the diagnostic tests.