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Changes in concanavalin A-mediated agglutination of hormone-dependent mouse mammary tumour cells during serial transplantation.

The concanavalin A-mediated agglutinability of GR mouse mammary tumour cells changes during serial transplantation of the tumours. Hormone-dependent cells in general have a lower agglutinability than hormone-independent cells. However, changes have also been observed in the histology of the tumours during serial transplantation, which as such may also alter the Con A-mediated agglutinability of the tumour cells.

Agglutination↗

Differential agglutination of particulate Vi and O antigens by the IgM and IgG class antibodies.

The IgG and IgM immunoglobulins were fractionated from pooled sera of typhoid patients and typhoid carriers and the agglutinin titres and weights of Vi and O antibodies in these immunoglobulin fractions determined. The O antigen particles were more effectively agglutinated than the Vi antigen particles by their corresponding antibodies of the IgM class. There was no such difference between IgG class Vi and O antibodies. Similar behaviour was found on indirect estimation of the agglutination capacity of the IgG and IgM class Vi and O antibodies in the sera of individual typhoid patients or carriers. The significance of such differential agglutinating capacity of the Vi and O antibodies of the IgM class is discussed.

Agglutination↗

Improving agglutination tests by working in microfluidic channels.

Latex agglutination tests are used for the diagnosis of diseases in man and animals. They are generally simple, cheap, and do not require sophisticated equipment, nor highly specialized skills. In this Technical Note, we put latex agglutination tests in a microfluidic format. The experiment is performed in PDMS (polydimethylsiloxane) microchannels, using streptavidin-coated superparamagnetic beads and a magnetic field. The target molecule is biotinylated protein A. By taking full advantage of the microfluidic conditions (scaling down of the detection volume and controlled action of the shear flow), we achieved an analytical sensitivity of 10 fmol l(-1)(several hundreds of fg ml(-1)) and a fast response (a few minutes) ; the test is also quantitative. Performances of agglutination tests can thus be improved by orders of magnitude by adapting them to a microfluidic format; this comes in addition to the usual advantages offered by this technology (integration, high throughput etc.).

Agglutination Tests↗

Effects of calcium ions and the bivalent cation ionophore A23187 on the agglutination and fusion of chicken erythrocytes by Sendai virus.

1. Ca2+ (0.4-16 mM) had no detectable action on the agglutination of hen erythrocytes by Sendai virus. 2. Pretreatment of the cells with Ca2+ (0.1-8 mM) in the presence of the bivalent cation ionophore A23187 led, however, to a significant decrease in the subsequent agglutination of the cells by the virus. 3. It thus appears that the entry of Ca2+ into the interior of these cells decreases cellular agglutination by Sendai virus; possible interpretations of this phenomenon are discussed in terms of the movement of intramembranous particles. 4. With a small number of virions, maximum cell fusion by Sendai virus occurred in the presence of EGTA [ethanedioxybis(ethylamine)tetra-acetate]. 5. Virus-induced cell fusion was significantly decreased by Ca2+, even at a concentration of 0.2 mM; it is suggested that this may result from diminished interactions between virus particles and erythrocyte membranes.

Agglutination↗

Purification of oncornaviruses by agglutination with concanacalin A (murine leukemia virus-phytohemagglutinin-friend virus).

Concanavalin A (Con A) has been used to rapidly and selectively agglutinate murine and avian oncornavirions from culture medium or plasma. The agglutinated virus was concentrated rapidly and gently by low-speed centrifugation and solubilization with alpha-methyl mannoside. Infectious virus was purified 2.3 times with respect to nucleic-acid content, and more than 60% of its infectivity was recovered. Infectious particles of densities 1.18 and 1.16 g/cm(3) were found in mouse cells infected with Friend virus. Con A reacted only with particles of density 1.16 g/cm(3), indicating heterogeneity with respect to carbohydrate content or structure as well as buoyant density. Electron microscopy of virus agglutinated with Con A showed a zone of Con A-glycoprotein complexes averaging 12-15 nm in thickness.

Agglutination↗

Correlation between the mobility of inner plasma membrane structure and agglutination by concanavalin A in two cell lines of MOPC 173 plasmocytoma cells.

Both the distribution of the concanavalin A-binding sites and the rearrangement of the intramembranous particles revealed by the freeze-etching technique, have been studied by means of two variants of the same cell line issued from MOPC 173 murine plasmocytoma. One variant does not agglutinate even in presence of high lectin concentration. It has been shown that the number of binding sites and affinity are almost the same in the two variants. The clustered distribution of intramembranous particles is induced by the interaction of the concanavalin A and the cell surface only in the variant which is agglutinable. From these results it became apparent that the clustered distribution of the membrane particulate components is an acquired feature of the plasma membrane accompanying cell agglutination.

Agglutination↗

The direct agglutination test: a non-specific test specific for the diagnosis of visceral leishmaniasis?

Serology has an important role to play in the diagnosis of the severe clinical syndrome of visceral leishmaniasis (VL). The direct agglutination test (DAT), a simple agglutination test which requires no laboratory facilities, has become the preferred test, particularly for field studies. The nature of the antigens responsible for the agglutination of leishmanial promastigotes by the serum of VL patients is not known. A series of experiments which provide some clues to the molecular basis for the test and which indicate that there might be more in DAT than meets the eye is reported.

Agglutination Tests↗

An analysis of lectin-initiated cell agglutination in a series of CHO subclones which respond morphologically to growth in dibutyryl cyclic AMP.

We have investigated the molecular basis of the agglutinability of CHO subclones which respond differentially in terms of morphology and surface architecture in the presence of dB-cAMP in the medium. We have demonstrated that the agglutinability of these subclones with both wheat germ agglutinin (WGA) and concanavalin A (Con A) probably depends on the free lateral mobility of the lectin receptor sites in the plane of the membrane. The nonagglutinable surface architecture seems to depend on the presence in the membrane of a protease-labile peptide(s), which appears to be distinct from the lectin receptors, as well as on continuous protein and RNA synthesis. This dependence on continuous transcription and translation may be related to the maintenance of the protease-labile peptide(s) in such a state as to restrict mobility of the lectin receptors. The surface architecture defined as nonagglutinable also depends on the state of polymerization of the intracellular microtubules and microfilaments. It is suggested that these microskeletal elements serve to anchor the lectin receptors in such a manner as to restrict their mobility and thereby reduce the relative agglutinability of a cell line. We suggest that control of the free mobility of both the Con A and WGA receptor sites is dependent on two constraints, one applied by protease-labile ("surface") membrane components and the other by components of the intracellular microskeletal system.

Agglutination↗

Surface properties related to concanavalin A-induced agglutination. A comparative study of several Entamoeba strains.

Pathogenic strains of Entamoeba histolytica are more easily agglutinated with concanavalin A (Con A) than strains isolated from human asymptomatic carriers. All three pathogenic strains studied here were found to agglutinate with low concentrations of Con A in contrast to various nonpathogenic axenic strains of amebas, characterized by their ability to grow at room temperature. Our present observations suggest that the extreme susceptibility of pathogenic strains of E. histolytica to agglutinate with Con A is related to their higher capacity for lectin binding and to their lack of detectable repulsive charges at the cell surface. The amount of fluorescein-tagged Con A bound to the surface was much higher in pathogenic strains. Only nonpathogenic strains showed a detectable negative surface charge as studied both by means of cell microelectrophoresis and by labeling cells with cationized ferritin at 0 degrees C. The mobility of surface Con A receptors estimated as the percentage of caps was comparable in all strains. Results of one strain cultured in axenic and monoxenic conditions suggested that bacteria can modify the behaviour of E. histolytica trophozoites by altering surface properties of the amebas.

Agglutination Tests↗

Plasmodium falciparum-infected erythrocytes: agglutination by diverse Kenyan plasma is associated with severe disease and young host age.

The variant surface antigens (VSAs) of Plasmodium falciparum-infected red blood cells are potentially important targets of naturally acquired immunity to malaria. Natural infections induce agglutinating antibodies specific to the VSA variants expressed by the infecting parasites. Previously, when different parasite isolates were tested against a panel of heterologous plasma from Kenyan children, the proportion of plasma that agglutinated the parasites (the agglutination frequency [AF]) was highly variable among isolates, suggesting the existence of rare and prevalent variants. Here, the AF of 115 isolates from Kenyan children were compared. The results show that the AF of isolates causing severe malaria were significantly higher than those of isolates causing mild malaria; and AF decreased significantly with the increasing age of the infected child. We propose that parasites causing severe disease tend to express a subset of VSA variants that are preferentially associated with infections of children with low immunity.

Agglutination Tests↗

Agglutination by concanavalin A of urothelial cells of heterotropically transplanted rat urinary bladders: effect of bladder carcinogens and urine.

N-butyl-N-(3-carboxypropyl)nitrosamine (BCPN) has been considered to be a carcinogenic urinary metabolite of N-butyl-N-(4-hydroxybutyl)nitrosamine. No tumor developed, however, in the heterotropically transplanted rat urinary bladders (HTBs) following repeated instillation of BCPN dissolved in physiological saline. In the present study, the possibility that BCPN dissolved in urine may induce tumors was explored using a short-term screening assay. When tested with the concanavalin A agglutination assay with which a close correlation between increase in cell agglutinability and carcinogenicity of test compounds has been well demonstrated, no significant increase in agglutinability attributable solely to BCPN was observed in HTB cells whether it was dissolved in saline or urine. Based on the current findings together with other available data, it is suggested that urothelial cells have a very limited capability to activate BCPN to the ultimate carcinogen, and require continuous contact with the carcinogen to respond with tumor formation.

Agglutination↗

Cloning of a glucose phosphate isomerase/neuroleukin-like sperm antigen involved in sperm agglutination.

The mouse monoclonal antibody (mAb) A36 produced by us and shown to induce extensive, "tangled" sperm agglutination was used to isolate cDNAs encoding its cognate antigen. Three overlapping cDNA clones specifically recognized by the mAb were isolated from a human testis cDNA expression library in lambdagt11. Sequencing of these cDNAs yielded the complete nucleotide sequence of a 3-kilobase cDNA that encodes the mAb-related polypeptide, designated sperm antigen-36 (SA-36), composed of 558 deduced amino acids. SA-36 cDNA contained a 5' untranslated region of 234 nucleotides (nt), an open reading frame of 1674 nt, and a 3' untranslated region of 1138 nt. SA-36 cDNA displayed > 99% homology to glucose phosphate isomerase (GPI)/neuroleukin (NLK) mRNA. This surprising homology was confirmed in Western blots demonstrating that mAb A36 reacted specifically with GPI obtained from rabbit muscle and from baker's yeast. Moreover, polyclonal, monospecific antibodies produced against beta-galactosidase/SA-36-3 fusion protein stained human spermatozoa and caused intensive agglutination of these cells in a manner similar to that with the mAb. Taken together, the data presented here demonstrated that mAb A36 cognate sperm surface antigen, encoded by SA-36 cDNA, is a GPI/NLK-like protein involved in sperm agglutination.

Amino Acid Sequence↗

Agglutination of blastospores of Candida albicans by concanavalin A and its relationship with the distribution of mannan polymers and the ultrastructure of the cell wall.

Blastospores of Candida albicans were readily agglutinated by Concanavalin A (Con A) owing to the specific binding of this lectin to the mannan receptors of the cell surface. When mannan was extracted from the cell wall by neutral buffers, alkali and acid, the agglutination was decreased or lost depending on the degree of extraction. A relatively mild alkali treatment was sufficient to derange the multilayered wall organization and transform it into a uniform, medium-density structure having about the same thickness as the untreated wall. After a more drastic extraction, all the electron-dense components of the wall were lost, the residual, alkali-insoluble wall fabric being completely electron-transparent and of about the same thickness as the inner wall region of untreated cells. Thiol-reducing agents like mercaptoethanol or dithiothreitol also extracted wall materials, an effect which was enhanced by pronase. After dithiothreitol-pronase treatment, the outer wall layers were removed but the inner wall region was not apparently damaged and some electron-dense components remained. None of these treatments significantly affected blastospore agglutination by Con A--this was reduced (but not abolished) only by the sequential action of pronase and helicase, which led to sphaeroplast formation. These sphaeroplasts showed a varied amount of residual wall consisting of evenly distributed, fibrogranular components. Two main conclusions were drawn from these results: (i) mannan polymers extend throughout the wall of the blastospore of C. albicans; (ii) the layering of the wall, as seen by ordinary fixation and staining for electron microscopy, essentially reflects the distribution of the various alkali-soluble complexes, at different levels, both over and in the rigid, glucan-chitin matrix.

Agglutination↗

The specificity of agglutination reactions of Pseudomonas aeruginosa with O antisera.

Polyagglutinable (PA) strains of Pseudomonas aeruginosa are agglutinated by more than one of the antisera prepared against antigenically unrelated O serogroups. They form c. 4% of strains--other than isolates from cystic-fibrosis units--submitted to us for typing. We were able to allocate 80% of PA strains to a single O serogroup by agglutination with typing sera that had been absorbed with the polyagglutinable strain SMC-247, or by co-agglutination tests with protein A-containing staphylococci coated with immunoglobulin from unabsorbed sera. Similar results were obtained by precipitation tests with crude bacterial extracts and unabsorbed sera, but these tests were less sensitive and less specific. Evidence is presented that PA antigen is a heat-stable cell constituent distinct from the O antigen. In rabbit antisera, anti-PA antibody is exclusively of the IgM class, but O antibody of both IgM and IgG classes is present.

Agglutination Tests↗

Serotyping and subtyping of Neisseria meningitidis isolates by co-agglutination, dot-blotting and ELISA.

Typing of meningococci with a panel of serotype and subtype specific monoclonal antibodies (MAbs) was compared in co-agglutination, dot-blotting and ELISA tests. Twenty reference strains, 50 case isolates and 133 throat isolates from healthy carriers were studied. The typing results with dot-blotting and ELISA were identical, whereas co-agglutination gave different results for three case and 24 carrier strains. The distribution of serotypes and subtypes among the strains is reported. The combination of the subtypes P1.1 and P1.15 in a serotype 15 patient strain was observed. With one case strain and 15 carrier strains, neither serotype nor subtype could be determined. Non-typable and non-subtypable isolates were further characterised by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Co-agglutination is useful for typing small numbers of strains with a few MAbs, but less suitable for large-scale typing than the other two methods. Dot-blotting needs less expensive equipment, smaller volumes of antibodies and fewer manipulations than ELISA.

Agglutination↗

Comparison of three microtube column agglutination systems for antibody screening: DG Gel, DiaMed-ID and Ortho BioVue.

The aims of the present study were to evaluate the estimated diagnostic accuracy of a new microtube column agglutination system (DG Gel, Diagnostic Grifols, Barcelona, Spain), to analyse the antibody reactivity and to compare the data with the two well-established DiaMed-ID and Ortho BioVue systems. We collected 3024 consecutive samples from blood donors, transfusion recipients and pregnant women, and 100 samples containing antibodies of known specificity. All these samples were tested in parallel by the three microtube agglutination systems. The estimated sensitivity was 100% for DG Gel and Ortho BioVue and 97.58% for DiaMed-ID. The estimated specificity was 99.93% for Ortho BioVue and 100% for DiaMed-ID and DG Gel. The score mean and range of the antibody titration of DG Gel, DiaMed-ID and Ortho BioVue were 34.31 (5-119), 30.3 (3-121) and 37.38 (3-112), respectively. All three column agglutination systems work well showing a high estimated diagnostic accuracy.

Agglutination Tests↗

Characteristics of an antibody causing agglutination of M-positive non-enzymatically glycosylated human red cells.

An alloagglutinin was identified in the serum of an M-negative diabetic patient. The agglutinin reacted with all commercial M-positive red cell samples. Routine crossmatches showed no incompatibility. This anti-M would only agglutinate M-positive red cell samples that had been incubated in 2% glucose for a minimum of 2 h at 37 degrees C, 2 days at 22 degrees C, or 1 week at 4 degrees C. Reactive red cell samples, when washed and incubated in saline, gradually became non-reactive. This antibody reacted optimally in low ionic strength solution at 16 degrees C for 20 min where MM red cells were agglutinated to a titer of 256, score 85; and MN red cells were agglutinated to a titer of 128, score 66. The antibody was denatured by 2-mercaptoethanol and was inhibited by a crude M tryptic isolate and by 2% glucose, but not by other sugars prepared at a 2% concentration.

Adult↗

Lectin agglutinability of mammary tumours with differing metastatic colonisation potentials.

A new fully quantitative method for assessment of lectin agglutinability has been used in this investigation to compare the surface composition of cells from tumours with high and low pulmonary colonisation potential. As in our previous work, we have used only primary (i.e., naturally-occurring) mammary tumours in mice. It was found that agglutinability with the lectins Concanavalin-A and Wheatgerm agglutinin bore no relationship to the pulmonary colonisation potential of the primary mammary tumour. However, cells from disaggregated secondary deposits of tumours which manifested high colonisation potential were consistently less agglutinable than the cells of the primary tumours from which they were derived.

Agglutination↗