Single laser three color immunofluorescence staining procedures based on energy transfer between phycoerythrin and cyanine 5.

Monoclonal antibodies specific for phycoerythrin (PE) were covalently labeled with the fluorescent dye cyanine 5 (Cy5). Excitation at 488 nm of immune complexes obtained by mixing Cy5-anti-PE with PE resulted in a 4-fold reduction of PE fluorescence measured at 565 nm and an increase of fluorescence measured at 655 nm. The observed energy transfer between PE and Cy5-anti-PE was used to develop three color immunofluorescence staining procedures for flow cytometers equipped with an Argon laser tuned at 488 nm. Mouse IgG1 monoclonal antibodies specific for cell surface antigens were cross-linked with either unlabeled or Cy5 labeled mouse IgG1 anti-PE using F(ab')2 fragments of monoclonal rat anti-mouse IgG1. PE was added to these immune complexes in sufficient amounts to saturate all PE binding sites. Cells were incubated with PE-labeled and PE/Cy5-labeled tetrameric antibody complexes together with FITC labeled antibodies and analyzed by flow cytometry. The emission from FITC, PE and PE/Cy5 could be readily separated and bright three color immunofluorescence staining of mononuclear cells from human peripheral blood and bone marrow was observed. The results of these experiments demonstrate that useful probes for single laser three color staining of cell surface antigens can be readily obtained by mixing of selected reagents. Compared to standard procedures for the covalent labeling of PE (tandem) molecules to antibodies, the non-covalent procedures described in this report provide significant advantages in terms of the amount of reagents, time and equipment required to obtain suitable reagents for three color immunofluorescence staining.     

Direct fluorescent staining and analysis of proteins on microspheres using CBQCA.

BACKGROUND: General methods for accurate determination of microsphere surface protein loading are needed for applications from protein arrays to molecular assembly studies. Current methods include bulk absorption measurements of stained microspheres or use of known fluorescently tagged binding partners, which limit sensitivity and general applicability, respectively. METHODS: Microspheres bearing covalently coupled proteins were stained with 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA) using different incubation times and dye concentrations to determine optimal staining conditions. The CBQCA fluorescence of microspheres (measured by flow cytometry) bearing known amounts of protein were used to generate standard curves of CBQCA fluorescence response versus the amount of microsphere surface protein. CBQCA was also used to stain noncovalent protein interactions. RESULTS: Maximal labeling was attained within 1 h with 1 mM CBQCA. Linear fluorescence response occurred between 8 x 10(4) and 1 x 10(6) proteins/microsphere. CBQCA staining did not disturb noncovalent protein interactions. CONCLUSIONS: We have developed methods using CBQCA and flow cytometry to quickly and simply quantify the amount of protein on the surface of a microsphere. Importantly, this approach could be extended to other formats (e.g., chips). Further, because it does not disturb noncovalent protein-protein interactions, it may be possible to use this approach to detect protein interactions without the use of purified prestained probes.     

Effective lattice behavior of fluorescence energy transfer at lamellar macromolecular interfaces.

Fluorescence energy transfer between donors and acceptors confined to macromolecular interfaces is considered. In particular, we discuss two theoretical models for the ensemble-average fluorescence intensity decay of the donor when both fluorophores are incorporated into a planar (e.g., lamellar) interface. The first model is based on a continuous distribution of donor and acceptor molecules on a two-dimensional surface, whereas the other assumes a discrete distribution of fluorophores along the nodes of a two-dimensional square lattice. Results for the discrete model show that the fluorescence intensity kinetics of a donor depends strongly on the geometry of the molecular distribution (i.e., the lattice constant) and the photophysics of fluorophores (i.e., critical radius of the energy transfer). Furthermore, a "discrete molecular distribution" might manifest itself in the experimental data as an increase in the apparent dimensionality of the energy transfer with increasing acceptor concentration. Altogether, the experimental and theoretical underpinnings indicate the enormous potential of using fluorescence energy-transfer kinetics for revealing structural features of molecular ensembles (i.e., geometry, shape) based on a single experimental measurement. However, further understanding the effects of restricted geometries on the fluorescence energy transfer is required to take full advantage of this information. Basic theoretical considerations to that end are provided.     

Analysis of antiphotobleaching reagents for use with FluoroNanogold in correlative microscopy.

Correlative microscopy is an important approach for bridging the resolution gap between fluorescence and electron microscopy. We have employed FluoroNanogold (FNG) as the detection system in these types of studies. This immunoprobe consists of a gold cluster compound to which a fluorochrome-labeled antibody is covalently linked. In these preparations, the fluorescence signal from FNG is first recorded then the gold cluster compound is subjected to a silver enhancement reaction before examination by electron microscopy. Potential complications are those associated with photochemical reactions that occur during fluorescence microscopy. We have evaluated this and some anti-photobleaching agents (i.e., 1,4-diazabicyclo[2.2.2]octane [DABCO],p-phenylenediamine [PPD], and N-propyl gallate [NPG]) for their utility with FNG in correlative microscopy. When DABCO was employed, the gold signal from FNG was dramatically diminished but the fluorescence signal was unaffected. The gold signal of DABCO-treated samples decreased to approximately 30% of that of the other samples. On the other hand, PPD and NPG did not adversely affect the FNG labeling. We recommend that either PPD or NPG be used and that DABCO be avoided as an antiphotobleaching reagent for this technique.     

Fringe-scan flow cytometry

We describe the development of a scanning flow cytometer capable of measuring the distribution of fluorescent dye along objects with a spatial resolution of 0.7 micron. The heart of this instrument, called a fringe-scan flow cytometer, is an interference field (i.e., a series of intense planes of illumination) produced by the intersection of two laser beams. Fluorescence profiles (i.e., records showing the intensity of fluorescence measured at 20 ns intervals) are recorded during the passage of objects through the fringe field. The shape of the fringe field is determined by recording light scatter profiles as 0.25 micron diameter microspheres traverse the field. The distribution of the fluorescent dye along each object passing through the fringe field is estimated from the recorded fluorescence profile using Fourier deconvolution. We show that the distribution of fluorescent dye along microsphere doublets and along propidium iodide stained human chromosomes can be determined accurately using fringe-scan flow cytometry. The accuracy of fringe-scan shape analysis was determined by comparing fluorescence profiles estimated from fringe-scan profiles for microspheres and chromosomes with fluorescence profiles for the same objects measured using slit-scan flow cytometry.     

Fluorescent nanometer microspheres as a reporter for sensitive detection of simulants of biological threats using multiplexed suspension arrays

We succeeded in using 40 nm FRET (fluorescence resonance energy transfer) microspheres conjugated to antibodies as the fluorescent reporters to perform the multiplexing suspension array measurements on two simulants of biological threats, ricin (A chain) and a crude spore preparation of Bacillus globigii (Bg). The microspheres were impregnated with two types of fluorophores in equal number (approximately 140 fluorophores in total per microsphere) and displayed bright PE-like fluorescence via a fluorescence resonance energy transfer mechanism. Activated microspheres (aldehyde groups) were directly coupled to antibodies and used to form sandwich-type immunoassays in a suspension array. For the crude preparations of Bg, the assay sensitivity using antibody-conjugated microspheres is an order of magnitude higher than that using the conventional fluorescent reporter, R-phycoerythrin (PE). Using the microspheres, Bg at the concentration of 5 ng/mL can be easily detected. For ricin, the assay sensitivity was similar to that obtained using PE as the reporter, but washing the reaction mixtures resulted in the fluorescence signals that were 2-3 times higher compared to those using PE. Ricin at a concentration of 1 ng/mL can be readily identified. Importantly, the two simulants do not interfere with each other in the multiplexing experiments. The 40 nm FRET microspheres are a new sensitive alternative as fluorescent reporters for detection in suspension arrays.     

Dye-incorporated poly(EGDMA-HEMA) microspheres as specific sorbents for aluminum removal

Aluminum [Al(III)] adsorption onto dye-incorporated poly(ethylene glycol dimethacrylate-hydroxyethyl methacrylate) [poly(EGDMA-HEMA)] microspheres was investigated. Poly(EGDMA-HEMA) microspheres, in the size range of 150–200 μm, were produced by a modified suspension polymerization of EGDMA and HEMA. The reactive dyes (i.e., Congo Red, Cibacron Blue F3GA and Alkali Blue 6B) were covalently incorporated to the microspheres. The maximum dye load was 14.5 μmol Congo Red/g, 16.5 μmol Cibacron Blue F3GA/g and 23.7 μmol Alkali Blue 6B/g polymer. The maximum Al(III) adsorption on the dye microspheres from aqueous solutions containing different amounts of Al(III) ions were 27.9 mg/g, 17.3 mg/g and 12.2 mg/g polymer for the Congo Red, Cibacron Blue F3GA and Alkali Blue 6B, respectively. The maximum Al(III) adsorption was observed at pH 7.0 in all cases. Non-specific Al(III) adsorption was about 0.84 mg/g polymer under the same conditions. High desorption ratios (95%) were achieved in all cases by using 0.1 M HNO₃. It was possible to reuse these dye-incorporated poly(EGDMA-HEMA) microspheres without significant losses in the Al(III) adsorption capacities.     

Synthesis of new hydrophilic microspheres: optimized carriers for microparticle-enhanced nephelometric immunoassays.

To optimize antigen-antibody reactions, we have synthesized chemically well-defined hydrophilic microspheres. Proteins or haptens were covalently linked to these carriers. When the microsphere conjugates were agglutinated by the corresponding antiserum, the size of the complex artificially increased during the immunological reaction. After optimizing various parameters such as the hydrophilic character, repulsion charges, and amount of antigen coupled to the microspheres, we developed a rapid and sensitive immunoassay based on laser light scattering by the complexes.     

Macrophage recognition of immune complexes: development and application of novel cell surface labeling procedures

A fluorescein- and lactoperoxidase-conjugated ferritin-anti-ferritin immune complex has been prepared for cell surface labeling experiments on immune recognition and effector function. Lactoperoxidase (LPO) has been covalently coupled to affinity-purified anti-ferritin antibodies with p-benzoquinone by a modified version of the method of Ternynck and Avrameas [Ternynck, T., & Avrameas, S. (1976) Ann. Immunol. (Paris) 127C, 197]. The conjugate is a heterodimer of Mr230 000 with linkages to either or both of the heavy and light chains of the antibody, as judged by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the absence and presence of 2-mercaptoethanol. The conjugate retains antibody-binding activity as measured by a quantitative precipitin assay. When incorporated into immune complexes, the modified antibody also retains Fc receptor recognition ability as determined by erythrocyte-antibody rosette inhibition assays. Electron microscopy demonstrated that the antigen, ferritin, was monodisperse with complete apoprotein sheaths surrounding the core. Ferritin-anti-ferritin-LPO complexes were formed in 4-fold antigen excess. Complexes were verified by fluorescence and electron microscopy. Immune complexes were masked with "cold" iodine by use of the endogenous LPO activity. The complexes bound to cells at 4 degrees C as shown by electron microscopy and fluorescence video/intensification microscopy. The LPO delivered to the cell surface in this fashion can be utilized to iodinate the surface with 125I. Under saturation conditions, the labeling with local LPO delivery followed by SDS-PAGE and autoradiography is identical with labeling with free LPO. Labeling has also been conducted under conditions of substrate deficit.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescence studies of dehydroergosterol in phosphatidylethanolamine/phosphatidylcholine bilayers

Our previous fluorescence study has provided indirect evidence that lipid headgroup components tend to adopt regular, superlattice-like lateral distribution in fluid phosphatidylethanolamine/phosphatidylcholine (PE/PC) bilayers (, Biophys. J. 73:1967-1976). Here we have further studied this intriguing phenomenon by making use of the fluorescence properties of a sterol probe, dehydroergosterol (DHE). Fluorescence emission spectra, fluorescence anisotropy (r), and time-resolved fluorescence intensity decays of DHE in 1-palmitoyl-2-oleoyl-PC (POPC)/1-palmitoyl-2-oleoyl-PE (POPE) mixtures were measured as a function of POPE mole fraction (X(PE)) at 23 degrees C. Deviations, including dips or kinks, in the ratio of fluorescence peak intensity at 375 nm/fluorescence peak intensity at 390 nm (I(375)/I(390)), fluorescence decay lifetime (tau), or rotational correlation time (rho) of DHE versus PE composition plots were found at X(PE) approximately 0.10, 0.25, 0.33, 0.65, 0.75, and 0.88. The critical values at X(PE) approximately 0.33 and 0.65 were consistently observed for all measured parameters. In addition, the locations, but not the depth, of the dips for X(PE) < 0.50 did not vary significantly over 10 days of annealing at 23 degrees C. The observed critical values of X(PE) coincide (within +/-0.03) with some of the critical mole fractions predicted by a headgroup superlattice model proposing that the PE and PC headgroups tend to be regularly distributed in the plane of the bilayer. These results agree favorably with those obtained in our previous fluorescence study using dipyrenylPC and Laurdan probes and thus support the proposition that 1) regular arrangement within a domain exists in fluid PE/PC bilayers, and 2) superlattice formation may play a significant role in controlling the lipid composition of cellular membranes (, Proc. Natl. Acad. Sci. USA. 95:4964-4969). The present data provide new information on the physical properties of such superlattice domains, i.e., the dielectric environment and rotational motion of membrane sterols appear to change abruptly as the lipid headgroups exhibit regular superlattice-like distributions in fluid bilayers.     

High-temperature fluorescent in situ hybridization for detecting Escherichia coli in seawater samples, using rRNA-targeted oligonucleotide probes and flow cytometry

Fluorescence in situ hybridization (FISH) is a widely used method to detect environmental microorganisms. The standard protocol is typically conducted at a temperature of 46 degrees C and a hybridization time of 2 or 3 h, using the fluorescence signal intensity as the sole parameter to evaluate the performance of FISH. This paper reports our results for optimizing the conditions of FISH using rRNA-targeted oligonucleotide probes and flow cytometry and the application of these protocols to the detection of Escherichia coli in seawater spiked with E.coli culture. We obtained two types of optimized protocols for FISH, which showed rapid results with a hybridization time of less than 30 min, with performance equivalent to or better than the standard protocol in terms of the fluorescence signal intensity and the FISH hybridization efficiency (i.e., the percentage of hybridized cells giving satisfactory fluorescence intensity): (i) one-step FISH (hybridization is conducted at 60 to 75 degrees C for 30 min) and (ii) two-step FISH (pretreatment in a 90 degrees C water bath for 5 min and a hybridizing step at 50 to 55 degrees C for 15 to 20 min). We also found that satisfactory fluorescence signal intensity does not necessarily guarantee satisfactory hybridization efficiency and the tightness of the targeted population when analyzed with a flow cytometer. We subsequently successfully applied the optimized protocols to E. coli-spiked seawater samples, i.e., obtained flow cytometric signatures where the E. coli population was well separated from other particles carrying fluorescence from nonspecific binding to probes or from autofluorescence, and had a good recovery rate of the spiked E. coli cells (90%).     

Specificity and site of action of a mammary gland thioesterase which releases acyl moieties from thioester linkage to the fatty acid synthetase

The lactone (I) of 2-hydroxy-1-naphthaleneacetic acid was developed as a reagent for novel, highly efficient, covalent attachment of an excited-state proton transfer fluorescence probe (i.e. 2-naphthol) to protein amino groups. The lactone (I) was shown to react with amines faster than it was hydrolyzed. Reaction of the lactone (I) with bovine serum albumin (BSA) was faster than its reaction with corresponding concentrations of small organic amines, which suggested that the lactone (I) first adsorbed to BSA and subsequently reacted covalently; data are presented which suggest that this covalent binding occurs at a unique, single site on BSA (i.e., affinity labeling). Equilibrium studies involving instantaneous and time-dependent fluorescence changes were interpreted in terms of a 1:1 lactone:BSA labeling ratio at neutral pH. Steady-state fluorescence spectroscopy of the 1:1 lactone:BSA conjugate and of the conjugate of the lactone with glycine ethyl ester were recorded, compared and interpreted in terms of the microenvironment of the protein-bound fluorescence probe. Applications are suggested for use of this new and powerful procedure for study of the conformational changes and molecular interactions in other proteins.     

Characterization of antibody binding to three cancer-related antigens using flow cytometry and cell tracking velocimetry

Proper antibody labeling is a fundamental step in the positive selection/isolation of rare cancer cells using immunomagnetic cell separation technology. Using either a two-step or single-step labeling protocol, we examined a combination of six different antibodies specific for three different antigens (epithelial specific antigen, epithelial membrane antigen, and HER-2/Neu) on two different breast cancer cell lines (HCC1954 and MCF-7). When a two-step labeling protocol was used (i.e., anti-surface marker-fluoroscein-isothiocyanate [FITC] [primary Ab], anti-FITC magnetic colloid [secondary Ab]) saturation of the primary antibody was determined using fluorescence intensity measurements from flow cytometry (FCM). The saturation of the secondary antibody (or saturation of a single-step labeling) was determined using magnetophoretic mobility measurements from cell tracking velocimetry (CTV). When the maximum magnetophoretic mobility was the primary objective, our results demonstrate that the quantities necessary for antibody saturation with respect to fluorescence intensity were generally higher than those recommended by the manufacturer. The results demonstrate that magnetophoretic mobility varies depending on the types of cell lines, primary antibodies, and concentration of secondary magnetic colloid-conjugated antibody. It is concluded that saturation studies are a vital preparatory step in any separation method involving antibody labeling, especially those that require the specificity of rare cell detection.     

Injectable polymeric microspheres with X-ray visibility. Preparation,properties, and potential utility as new traceable bulking agents

The copolymer of methyl methacrylate (MMA) and 2-[2',3',5'-triiodobenzoyl]oxoethyl methacrylate (1), ratio 3:1 (mass:mass), was prepared via a free-radical polymerization in bulk. The copolymer (M(w) = 97.8 kD and M(n) = 41.5 kD) was dissolved in chloroform and subsequently transformed into beads with a diameter in the micrometer range, using a solvent evaporation technique. The resulting microbeads were characterized by different techniques, including NMR spectroscopy, differential scanning calorimetry, gel permeation chromatography, and scanning electron microscopy. The latter technique was used as the basis for statistical analysis of the bead size. Typically, an average diameter of 96 microm and a standard deviation of 21 microm were obtained. The beads were also subjected to some preliminary tests regarding cytotoxicity. The copolymer of MMA and 1 contains covalently bound iodine. Therefore, the material is intrinsically radiopaque, i.e., capable of absorbing X-radiation while no contrast additive is needed. Our interest in these microspheres stems primarily from their possible utility as injectable and afterward traceable (radiopaque) bulking agents, e.g., for use in urology for the treatment of female stress incontinence due to sphincter deficiency. As a first test into this direction, a sample of the microbeads was mixed with ethylene glycol, and the resulting suspension was studied with respect to injectability and radiopacity. The results suggest that the radiopaque microbeads may provide access to improved bulking agents. Further modification of the surface may be necessary in order to suppress the migratory aptitude of the radiopaque polymeric microspheres in vivo.     

Fluorescence energy transfer as a probe for nucleic acid structures and sequences.

The primary or secondary structure of single-stranded nucleic acids has been investigated with fluorescent oligonucleotides, i.e., oligonucleotides covalently linked to a fluorescent dye. Five different chromophores were used: 2-methoxy-6-chloro-9-amino-acridine, coumarin 500, fluorescein, rhodamine and ethidium. The chemical synthesis of derivatized oligonucleotides is described. Hybridization of two fluorescent oligonucleotides to adjacent nucleic acid sequences led to fluorescence excitation energy transfer between the donor and the acceptor dyes. This phenomenon was used to probe primary and secondary structures of DNA fragments and the orientation of oligodeoxynucleotides synthesized with the alpha-anomers of nucleoside units. Fluorescence energy transfer can be used to reveal the formation of hairpin structures and the translocation of genes between two chromosomes.     

Comparison of the Sequence-Dependent Fluorescence of the Cyanine Dyes Cy3, Cy5, DyLight DY547 and DyLight DY647 on Single-Stranded DNA

Cyanine dyes are commonly used for fluorescent labeling of DNA and RNA oligonucleotides in applications including qPCR, sequencing, fluorescence in situ hybridization, Förster resonance energy transfer, and labeling for microarray hybridization. Previous research has shown that the fluorescence efficiency of Cy3 and Cy5, covalently attached to the 5′ end of single-stranded DNA, is strongly sequence dependent. Here, we show that DY547 and DY647, two alternative cyanine dyes that are becoming widely used for nucleic acid labeling, have a similar pattern of sequence-dependence, with adjacent purines resulting in higher intensity and adjacent cytosines resulting in lower intensity. Investigated over the range of all 1024 possible DNA 5mers, the intensities of Cy3 and Cy5 drop by ∼50% and ∼65% with respect to their maxima, respectively, whereas the intensities of DY547 and DY647 fall by ∼45% and ∼40%, respectively. The reduced magnitude of change of the fluorescence intensity of the DyLight dyes, particularly of DY647 in comparison with Cy5, suggests that these dyes are less likely to introduce sequence-dependent bias into experiments based on fluorescent labeling of nucleic acids.     

The FDAM method: determination of carboxyl profiles in cellulosic materials by combining group-selective fluorescence labeling with GPC

A novel method for accurate determination of the carboxyl content in cellulosic materials by fluorescence labeling with 9H-fluoren-2-yl-diazomethane (FDAM) has been developed. The procedure can readily be implemented into a GPC system with RI and MALLS detectors, requiring additional fluorescence detection. The labeling conditions were optimized by means of sugar acid model compounds and were transferred to the cellulose case. Kinetics of the labeling and the influence of reaction parameters were comprehensively studied. For the first time, carboxyl profiles of cellulosics, i.e., the carboxyl content relative to the molecular weight distribution, were obtained.     

Automatic cell identification and enrichment in lung cancer: V. Adenocarcinoma and large cell undifferentiated carcinoma

The aims of this study were to develop a protocol for the identification and enrichment of cancer cells from sputum obtained from patients with adenocarcinoma of the lung (n = 6) and large-cell undifferentiated carcinoma of the lung (n = 2), and to compare these findings with the results from our previous studies on other cell types from lung cancer. The hypotheses tested were: Cancer cells in sputum can be preserved following flow sorting. Enrichment for cancer cells from acridine orange (AO)-stained specimens can be achieved. Discrimination of cancer cells from noncancer cells is by AO green fluorescence and discrimination of lymphocytes from other cell types is by AO red fluorescence. Cancer cells are consistently enriched in the AO high green and red fluorescence region, although, for a given cell type, maximal enrichment is patient-dependent. Finally, cancer cell enrichment and lymphocyte exclusion can be done simultaneously. Cells from sputum were initially fixed, stained with AO, sorted on a dual parameter flow sorter, and classified into six groups corresponding to two ranges of green and three ranges of red fluorescence intensities. Cells of each region were stained by the method of Papanicolaou and differential counts were performed to determine the relative frequencies (i.e., purities) of leukocytes, macrophages, squamous cells, and cancer cells, in sorted and unsorted (i.e., control) samples. The average purity of leukocytes (81%), macrophages (6%), squamous cells (11%), and cancer cells (2%) varied markedly from sample to sample. However, the largest enrichment values (i.e., ratio of purity of a cell type in a sorted sample to its purity in the unsorted control sample) achieved for cancer cells consistently occurred for each patient sample in the region corresponding to high green and high red fluorescence intensities. Experimentally, a cancer cell average enrichment of sixteen-fold was obtained by this method. Additionally, fluorescence intensity ranges which increased the enrichment for macrophages by cell sorting typically excluded leukocytes and squamous cells, and vice versa. Finally, red fluorescence intensity was the primary discriminatory parameter for all cell types studied, although the additional use of green fluorescence intensity significantly increased cancer cell enrichment rates.(ABSTRACT TRUNCATED AT 400 WORDS)     

Alterations of wheat-germ agglutinin binding pattern on cell surface of blood platelets after thrombin stimulation

An attempt was made to demonstrate wheat-germ agglutinin (WGA) binding sites on platelet surfaces after thrombin stimulation, by means of a post-embedding cytochemical technique using colloidal gold as marker at an ultrastructural level. In unstimulated platelets washed with EDTA, an intense uniform labeling of WGA-gold complexes was found on the surface membrane. When washed platelets were stimulated by thrombin in the absence of Ca2+, only a release reaction was induced. WGA labeling on the surface membranes of these platelets decreased dramatically. However, the labeling intensity of WGA-gold complexes on the surface membrane of aggregated platelets induced by thrombin in the presence of Ca2+ increased significantly compared to that of thrombin-stimulated platelets in the absence of Ca2+. In contrast to the uniform labeling on the surface membranes of unstimulated platelets, clusters of gold label were often found on the surface membrane of the aggregated platelets, although there was no significant quantitative difference in the labeling intensity between these two groups. Thus, we present direct morphological evidence demonstrating qualitative and quantitative alterations of WGA labeling on the surface membrane of platelets after thrombin stimulation. The possibility is considered that WGA-binding glycoproteins in the surface membrane are involved in the aggregation response after thrombin stimulation.     

Surface antigens of Biomphalaria glabrata (Gastropoda) hemocytes: evidence for linkage-independence of some hemolymph-like surface antigens and Con A receptor-bearing macromolecules

The structural relationship between hemolymph-like surface antigens and concanavalin A (Con A)-reactive macromolecules on circulating hemocytes of Biomphalaria glabrata was assessed using a double-ligand labeling method. It was determined that Con A-induced clearance of its own receptor complexes resulted in a significant reduction, but of complete elimination, of hemolymph-like antigens, i.e., antigens cross-reactive with an anti-B. glabrata hemolymph antiserum, suggesting the presence of at least two separate antigenic populations with anti-hemolymph reactivity; one group structurally linked and another group structurally independent of Con A-binding membrane components. The latter group of surface antigens appears to be (1) chemically related to the higher-molecular-weight hemolymph components, most probably hemoglobin, (2) Pronase resistant, and (3) partially composed of a subpopulation of cryptic (hidden) cross-reacting antigens uncovered at the cell surface as a consequence of Con A-receptor clearance. Results of this study demonstrate not only that individual hemocytes of B. glabrata may possess different populations of hemolymph-like antigens, but also that the interaction between some membrane components and appropriate ligands (e.g., carbohydrate-binding lectins) could result in a modulation in expression of other groups of surface antigens.