Extent Of Agglutination Research Articles

A Reliable von Willebrand Factor: Ristocetin Cofactor Enzyme-Linked Immunosorbent Assay to Differentiate between Type 1 and Type 2 von Willebrand Disease

von Willebrand disease (vWD) is the most common hereditary bleeding disorder due to either a qualitative or a quantitative defect in von Willebrand factor (vWF). vWF is a multimeric plasma protein that plays an important role in (1) primary hemostasis, by sustaining indirect platelet adhesion especially at high shear rates, and in (2) secondary hemostasis, by protecting factor VIIIc (FVIIIc) from degradation. A correct diagnosis of vWD is based on the accurate identification of one of the six different subtypes (type 1, 2A, 2B, 2M, 2N, 3). To do this, different laboratory tests are available. One aspect of the identification is the discrimination between type 1 and type 2 (2A, 2B, and 2M) vWD. In type 1 vWD, both vWF levels (vWF:Ag) and vWF activity (vWF:RCof) are decreased; in type 2, the vWF:Ag level is normal or decreased and vWF:RCof is decreased. Thus, ratios of vWF:Ag to vWF:RiCof above 1 allow identifation of type 2 vWD patients. The currently used vWF:RCof test is an agglutination test in which patients' plasma is added to washed fixed control platelets in the presence of ristocetin and the extent of agglutination is measured. This test suffers from high interlaboratory and intralaboratory variability. We have recently shown that the same vWF:RCof can also be measured in an enzyme-linked immunosorbent assay (ELISA) with a low interassay and intraassay variability and can be used to identify patients suffering from vWD. We here show that our test allows the discrimination between type 1 and type 2 vWD patients.

An agglutination assay using emulsified oils

Dispersed fluorocarbon oil droplets were used as the antigen-carrier particle in an immunological agglutination assay. The extent of agglutination was found to vary with the percentage of antigen to that of inert protein used in the solution to coat and stabilize the oil droplets. Maximum sensitivity was observed when 6.25% of the protein adsorbed on the oil droplets was specific antigen. At this percentage, antibodies were detected at concentrations as low as 0.10 μg/ml. Results were obtained by visual examination of the reacted samples as well as with quantitative computer analysis using the Zeiss IBAS image analysis system.

Secretory immunoglobulin binding to bacteria in the mouse uterus after mating

Bacteria of several species were present in the mouse uterus on the morning after mating, as demonstrated by bacteriol cultures and microscopic examination of Gram-stained uterine luminal contents. The similarity between bacteria cultured from the vagina before mating and from the uterus after mating suggested that bacteria were introduced into the uterus from the vagina, possibly by coitus. The bacteria were cleared from the uterus about two days after mating. Immunohistochemical labeling of smears of the luminal contents on the morning after mating demonstrated IgA, IgG, and possibly IgM bound to many of the bacteria. The bacteria were often agglutinated, and there was a correlation between the intensity of immunoglobulin labeling on bacteria and the extent of agglutination. The amount of antibody bound to bacteria in multiparous mice was about the same as in mice that had not been mated previously. We observed both IgG and IgA on bacteria when organisms from vaginal cultures were incubated for 60 min in the uteri of estrogen-primed, virgin, female mice. This indicated that the uterus was the source of at least part of the immunoglobulins bound to bacteria. We did not demonstrate that the immunoglobulins bound to bacteria were specific anti-bacterial antibodies, but the binding persisted through three washing steps and there wasno immunoglobulin binding to sperm in the same preparations. Neutrophils in the uterine lumen on the day after mating contained phagocytosed bacteria. These results suggest that the secretory immune system in the female mouse reproductive tract may play a role in returning the uterus to an aseptic state after mating by at least three mechanisms: direct blocking of attachment sites involved in bacterial binding to mucosal epithelium, agglutination of bacteria and thus reduction in the number of organisms available for binding to the epithelium, and opsonization of bacteria for phagocytosis by neutrophils.

Temperature dependence of ricinus communis agglutinin activity

Ricinus communis agglutinin, a galactose-binding lectin, agglutinates phospholipid/qlycolipid vesicles, phospholipid/glycolipid/cholesterol vesicles, low density lipoprotein, and the branched polysaccharide gum arabic. The temperature dependence of the initial velocity of agglutination is similar in all cases, with a maximum at ∼25°C, and a decreased rate at lower and higher temperatures. The extent of agglutination is temperature-independent for T ≤ 25°C, and decreases with increasing temperature for T > 25°C. After agglutination at 25°C, an increase in temperature results in deagglutination. This effect is reversible upon return to 25°C. Thus RCA undergoes a reversible temperature-dependent transition between an agglutinating form (≤25°C) and a less active form (> 25°C). These observations have important consequences in the interpretation of experiments which attempt to correlate lectin-binding with temperature-dependent properties such as membrane fluidity or receptor lateral organization in natural and model membranes.

Surface-associated host proteins on virulent Treponema pallidum.

A surface coat of host serum proteins was detected on virulent Treponema pallidum by sodium dodecyl sulfate-gel electrophoresis. The loosely associated serum proteins could be removed by repeated washings in a protein-free medium. Washed T. pallidum retained the ability to readsorb numerous host proteins from rabbit serum as well as iodinated rabbit or human albumin. In addition, various avidly associated host serum proteins including albumin, alpha(2)-macroglobulin, transferrin, ceruloplasmin, immunoglobulin G, immunoglobulin M, and C3 were identified on the outer envelope of washed treponemes by an immunoadsorbent technique with protein A-bearing staphylococcus. Hyaluronidase treatment did not remove the avidly associated host proteins from the surface of washed treponemes, whereas trypsin treatment resulted in decreased levels of agglutination. Electrophoretic patterns of trypsin-treated treponemes showed that treponemal proteins as well as adsorbed host proteins were released concurrently by protease digestion. Reacquisition studies involving alpha(2)-macroglobulin and transferrin suggested the presence of noncompetitive binding sites for serum proteins on the treponemal outer envelope. Finally, differences among the T. pallidum preparations from individual rabbits with respect to incorporation of [(35)S]methionine, extent of agglutination with antisera, and length of time required for removal of avidly associated host proteins by trypsin treatment indicated biological variability among the treponemal populations.

Species-specific sperm adhesion in sea urchins. A quantitative investigation of bindin- mediated egg agglutination

Bindin, a protein component of the acrosomal vesicle of sea urchin sperm, has been isolated from Arbacia punctulata and strongylocentrous purpuratus. Using this isolated bindin, we have devised a quantitative assay for bindin-mediated egg agglutination and compared the agglutination of bindin eggs from A. puntulata and S. purpuratus. Bindin- mediated agglutination is species -specific in both species, although a measurable degree of heterotypic interaction is observed. Homotypic bindin-egg interactions differ significantly from heterotypic interactions both in the extent of agglutination and the size of the resulting aggregates. We also provide direct evidence that bindin particles agglutinate eggs by adhering to the surfaces of adjacent eggs. Although the A. punctulata bindin preparation displays the same functional properties and consists of one major polypeptide of the same apparent molecular weight as S. purpuratus bindin, its morphology is very different. Unlike the spherical aggregates observed with S. purpuratus bindin, A punctulata bindin exists as lamellar vesicles and binds significant amounts of phospholipids and Triton X-100, suggesting that it may be tightly associated with the acrosomal membrane. Having defined a number of the basic parameters of bindin-mediated agglutination, we examined the effect of a number of saccharides and glycopeptides on bindin-mediated egg agglutination. Carbohydrate-containing components derived from the egg cell surface by proteolysis were found to inhibit bindin-mediated egg agglutination at low concentrations, but this inhibition is not species specific.

The role of flagellar adhesion in sexual activation ofChlamydomonas eugametos

Sex-specific agglutination in gametes ofChlamydomonas eugametos was carried out with dead partner cells (killed by glutaraldehyde, formaldehyde or OsO4), with isolated flagella, flagellar appendages and isoagglutinins derived from cell-free culture medium. The activation of the plasma papilla in agglutinated cells was studied by scanning electron microscopy in relation to the agglutinative properties of the materials tested. The results reveal differences in agglutination and papilla activation between gametes of both mating types. They also indicate that papilla activation depends on the extent of agglutination, which is a function of sex-specific flagellar properties and the amount and nature of the agglutinative material used. A hypothesis is presented to explain the observed phenomena.

The effect of cytochalasin B and colchicine on lectin-mediated agglutination of early embryonic chick cells.

Abstract The effects of cytochalasin B (CB) and colchicine on the lectin-mediated agglutination of dissociated cells from chick embryos at the early primitive streak stage were studied. Cells incubated in the absence of the above-mentioned drugs were agglutinable with concanavalin A (ConA), wheat germ agglutinin (WGA), and Ricinus communis agglutinin (RCA). A pre-incubation with neuraminidase was required to render the cells agglutinable with soybean agglutinin (SBA). This treatment had no appreciable effect on the agglutinability of the cells with the other three lectins. Treatment with the drug colchicine had no appreciable effect on the extent of agglutination with any of the above-mentioned lectins. Cells treated with CB dissolved in dimethylsulfoxide (DMSO) in saline, exhibited a reduced lectin-mediated agglutinability. However, a similar decline in agglutinability was observed in controls incubated in saline containing DMSO alone. It is suggested that structures sensitive to colchicine and CB do not play a major role in the control of surface lectin receptors in early embryonic cells.

The effect of temperature of concanavalin A-mediated agglutination of cells with rigid receptors

The agglutination of a yeast, Candida albicans, by concanavalin A has been described. The agglutination was cell-number dependent. Prolonged incubation (60 min) was needed to reach maximum agglutination at 37° C. The rate but not the extent of agglutination was temperature dependent. The dimeric forms of concanavalin A, obtained either at low pH or after succinylation, agglutinated the yeast cells as well as the tetramer. Temperature changes affected the agglutination of yeast cells by dimers and by tetramers to the same extent.

Studies on the interaction of dopamine β-hydroxylase from various sources with phytohaemagglutinins

1. 1. Bovine adrenal medulla dopamine β-hydroxylase, a glycoprotein with terminal mannose residues in the carbohydrate moiety, did not precipitate with any lectins tested except concanavalin A. After digestion with neuraminidase, the enzyme was shown to interact with ricin, and a good correlation was found between the amount of liberated sialic acids and the extent of agglutination. This finding shows either that more than one type of carbohydrate unit occurs on the protein or that three are multibranched chains in the carbohydrate moiety. 2. 2. Dopamine β-hydroxylase from human serum, pheochromocytoma and normal adrenal were incubated with concanavalin A, ricin, wheat germ agglutinin and lectins from Dilochos biflorus and Robinia pseudoacacia. 83% of dopamine β-hydroxylase from pheochromocytoma was precipitated by ricin, whereas the enzyme from human serum precipitated to a lesser extent (5–15%). Neuraminidase digestion of human serum dopamine β-hydroxylase led to an increase of precipitation with ricin. The low extent of native human serum dopamine β-hydroxylase precipitation with ricin can be explained by the attack of plasma membrane sialidases of liver cells, whereas the greater ricin precipitation of pheochromocytoma and normal adrenal dopamine β-hydroxylases could be due to post-mortem effects. The clinical implications of possibility of differences concerning the carbohydrate moiety structures of pheochromocytoma and normal enzymes is discussed.