Erythrocyte diagnostic kits for detection of Pseudomonas aeruginosa exotoxin A

The use of formulated chick red blood cells loaded with IgG preparations and affinity-purified antibodies, in comparison with initial immune serum to P. aeruginosa exotoxin A (ETA), has been shown to increase the sensitivity of antibody erythrocyte diagnosticum (AbED) 17-fold and to ensure the detection of ETA at a concentration of 1.2 mg of protein per ml. The passive hemagglutination (PHA) test with AbED has proved to be a more sensitive method for the detection of ETA than the antibody neutralization test with the use of antigenic erythrocyte diagnosticum, the latex agglutination test, the coagglutination test and the enzyme immunoassay. The PHA test has permitted the detection of ETA in the culture fluid of 80% of P. aeruginosa cultures under study.     

Latex immunoassay of human serum Lp(a+) lipoprotein

A sensitive latex immunoassay for human serum lipoprotein Lp(a+) is based on direct agglutination by Lp(a+) of latex particles coated with specific antibody. The agglutination is quantified by turbidimetry using a photometer at 360 nm. The stabilization of antibody-coated latex particles by bovine serum albumin occurs under well-defined conditions (pH, concentration of bovine serum albumin, and antibody loading of latex particles). The standard curve of serum lipoprotein Lp(a+) ranges from 0.05 to 1.15 mg/l. Inter- and intra-assay coefficients of variation were less than 8% and 3%, respectively. Results were well correlated with those obtained by electroimmunodiffusion (r = 0.98, n = 108).     

Conventional detection method of fibrinogen and fibrin degradation products using latex piezoelectric immunoassay

We developed a conventional immunosensor for fibrinogen and fibrin degradation products (FDP) to combine a quartz crystal microbalance (QCM) with the agglutination reaction of immunized latex beads. FDP induced an immunoreaction due to anti-FDP antibody immobilized latex particles. We successfully measured FDP concentration of in human serum within 10 min by QCM method. The detection range of QCM immunosensor is covered with screening concentration of FDP in serum (<10 microg/ml of FDP). The time course of latex agglutination obtained from QCM immunosensor is synchronized to that of latex photometric immunoassay. SEM was used to observe the surface of QCM that applied FDP serum. The size of latex particles agglutinated on the QCM electrode increased concomitant with FDP concentration. Frequency shift on immunoreaction explains the increased adsorption amount of agglutinated latex on QCM.     

Specificity of anti-human IgG antibodies in antiglobulin (Coombs) sera.

The agglutination test with latex particles coated with aggregated human IgG was introduced into the evaluation of Coombs serum as an additional test for anti-IgG antibody activity. In Coombs sera prepared by the conventional immunization method employing Freund's adjuvant, latex agglutination titers were found much lower than those of anti-D-coated red cell agglutination. On the other hand, in sera prepared by other immunization methods, such as the one according to Haynes and Chaplin (1971), anti-IgG antibody response was readily observed by IgG-coated latex agglutination. Specificity of anti-IgG antibodies in the latter sera seems to be predominantly directed to aggregated human IgG.     

Development and application of thermo-sensitive magnetic immunomicrospheres for antibody purification

Ultrafine magnetite particles were prepared by a co-precipitation method. The poly-(styrene/N-isopropylacrylamide/methacrylic acid) latex particles containing ultrafine magnetite [magnetic P(St/NIPAM/MAA)] were prepared by two-step emulsifier-free emulsion polymerization. The minimum NaCl concentration for flocculation of these magnetic latex particles (critical flocculation concentration, CFC) decreased with increasing temperature. These temperature dependence of CFC, namely its thermo-sensitivity, originated from NIPAM. At a certain NaCl concentration, some of the magnetic latex particles showed reversible transition between flocculation and dispersion by controlling the temperature, and the thermo-flocculated magnetic latex particles were separated quickly in a magnetic field. Bovine serum albumin (BSA) was covalently immobilized onto the magnetic P(St/NIPAM/MAA) latex particles with high efficiency by the carbodiimide method. These thermo-sensitive magnetic immunomicrospheres were effective for the immunoaffinity purification of anti-BSA antibodies from antiserum.Correspondence to: A. Kondo     

Rubber elongation factor from Hevea brasiliensis. Identification, characterization, and role in rubber biosynthesis

The presence of a protein, rubber elongation factor (REF), which is tightly bound to serum-free rubber particles purified from Hevea brasiliensis latex, is necessary for prenyltransferases from a number of sources to add multiple cis-isoprene units to rubber molecules. These prenyltransferases show normal farnesyl pyrophosphate synthase activity (two trans additions of isopentenyl pyrophosphate to dimethylallyl pyrophosphate) in the absence of REF bound to rubber particles. REF bound to rubber molecules can be highly purified from all other proteins in whole latex by treatment of rubber particles with low concentrations of detergent. Treatment of rubber particles with trypsin which hydrolyzes bound REF, removal of REF with high concentrations of various detergents, or treatment of whole latex with polyclonal antibodies specific for REF all prevent prenyltransferase from adding [14C]isopentenyl pyrophosphate to rubber molecules. However, we have not been successful using detergent-solubilized REF in the reconstitution of in vitro rubber biosynthesis with either REF-depleted rubber particles or allylic pyrophosphate primers. REF has a molecular mass of 14,600 Da and is associated specifically with rubber particles in whole latex. It makes up between 10-60% of the total protein in whole latex but is absent in C-serum, the supernatant fluid obtained when rubber particles are removed by centrifugation. The amount of REF in whole latex is proportional to the rubber content. Based on a number average molecular mass of 500,000 Da for rubber and the content of rubber and REF in whole latex or serum-free rubber particles, the stoichiometry of REF molecules to rubber molecules is 1:1 in both cases. There is sufficient REF to form a monomolecular protein layer coating large rubber particles (700-1,000 nm). In the electron microscope, serum-free rubber particle preparations contain particles with diameters from 800 to as small as 10 nm. In the presence of 1% sodium dodecyl sulfate no particles smaller than 100 nm are observed. We suggest that the smaller particles may be mainly composed of REF molecules.     

A novel approach to nonradioactive hybridization assay of nucleic acids using stained latex particles.

The paper describes a sensitive latex hybridization assay (LHA) method applied for indirect detection of biotinylated nucleic acid hybrids immobilized on a synthetic membrane. The biotinylated hybrids were visualized by means of latex particles containing the fluorescent dye pyronine G and coated with streptavidin; 1.6 and 0.3 pg of lambda-phage DNA was detected by dot blot hybridizations on nylon membrane and polyethyleneimine-cellophane, respectively. The assay sensitivity was increased by three orders of magnitude over that with fluorescently labeled probes due to encapsulation of the fluorescent dye in polymer particles. LHA is simple (single-stage detection procedure), fast, and more sensitive than any of the other nonradioactive hybridization methods.     

Rapid latex agglutination test for detection of staphylococcal enterotoxins A to E that uses high-density latex particles.

A rapid reversed passive latex agglutination method that uses high-density latex particles for the detection of staphylococcal enterotoxins (SE) A to E was developed. It took 3 h for incubation, much less than the 16 h needed with a customary latex agglutination test for SE detection such as a commercial test kit (SET-RPLA; Denka Seiken Co. Ltd., Tokyo, Japan). The rapid test was shown to be highly specific and sensitive for SE detection (detection limit, about 0.5 ng of SE per ml), comparable to the SET-RPLA test. The rapid test was also efficient in SE detection in foods and culture supernatants of staphylococcal strains, similar to the SET-RPLA test. This showed that a rapid test with high-density latex particles is fully reliable for use.     

Rapid latex agglutination test for detection of staphylococcal enterotoxins A to E that uses high-density latex particles

A rapid reversed passive latex agglutination method that uses high-density latex particles for the detection of staphylococcal enterotoxins (SE) A to E was developed. It took 3 h for incubation, much less than the 16 h needed with a customary latex agglutination test for SE detection such as a commercial test kit (SET-RPLA; Denka Seiken Co. Ltd., Tokyo, Japan). The rapid test was shown to be highly specific and sensitive for SE detection (detection limit, about 0.5 ng of SE per ml), comparable to the SET-RPLA test. The rapid test was also efficient in SE detection in foods and culture supernatants of staphylococcal strains, similar to the SET-RPLA test. This showed that a rapid test with high-density latex particles is fully reliable for use.     

A sensitive silver stain for proteins in agarose gels

A silver stain for proteins in agarose gels which is at least 10 times as sensitive as Coomassie blue is described. The method is simple to use and is particularly useful for the study of protein bands in the gamma region on electrophoresis of fluids such as cerebrospinal fluid in which the protein concentration is low. It readily detects bands of IgG containing 20 to 40 ng/band (approx 3 to 6 ng of IgG/mm2 of gel).     

Flow cytometric assay for phagocytosis of human monocytes mediated via Fc gamma-receptors and complement receptor CR1 (CD35).

We developed a simple flow cytometric assay for phagocytosis by human monocytes that is mediated via Fc gamma receptors and the complement receptor CR1 (CD35), using fluorescent latex beads carrying IgG and complement components C4b and C3b. To prepare fluorescent latex beads carrying IgG(BA), BSA-coated latex beads (B) were incubated with diluted rabbit anti-BSA IgG. To bind complement components, BA-particles were incubated with whole human serum pretreated with K-76 monocarboxylic acid (K-76COOH). K-76COOH inhibits the activities of C5 and factor I (12,13), resulting in the deposition of C1,4b,2a,3b on BA-particles (BAC1,4b,2a,3b). Further incubation of BAC1,4b,2a,3b with EDTA-GVB at 37 degrees C gave particles carrying IgG and C4b,C3b (BAC4b,3b). The C3 fragment, C3b, was confirmed to present on BAC1,4a,2a,3b particles by SDS-PAGE and immunoblot, and these particles were calculated to have approximately 25,000-30,000 C3b molecules per particle. To evaluate the particle attachment, the phagocytic assay was performed with 3 microM cytochalasin D treated cells. The percent cells with ingested particles and the number of ingested particles/100 cells for 60 min were estimated, being 5.1% and 5.4 for B, 12.3% and 26.7 for BA, 42.5% and 108.7 for BAC4b,3b, and 42.6% and 112.5 for BAC1,4b,2a,3b, respectively.     

Polarization of endocytosis and receptor topography on cultured macrophages

In the 1774.2 macrophage cell line, microtubule disassembly by colchicine causes the polarization of membrane functions ane structure. Colchicine-treated cells develop a bulge or protuberance that is bordered by microvillous membrane. The protuberance is the site of concanavalin A cap formation. The fluid pinocytosis of horseradish peroxidase and of fluorescein- and rhodamine-conjugated high molecular- weight dextrans, the adsorptive pinocytosis of concanavalin A, and the concentration and phagocytosis at 37 degrees C of a range of phagocytic particles (IgG- and complement-opsonized erythrocytes, complement- opsonized zymosan, latex shpres, albumin-stabilized oil droplets) are all similarly restricted to the protuberance. A reduction in the rate of dextran pinocytosis, determined by fluorimetry, and reductions in phagocytic rates for oil emulsion and IgG-opsonized erythrocytes accompany the polarization of endocytic activity in colchicine-trated 1774.2 macrophages. Membrane receptors for phagocytic particles are not confined to the protuberance but rather may display their own unique topographical asymmetry. The inherent topography of receptors was inferred from particle distribution under conditions that limit particle-receptor redistribution (after labeling at 4 degrees C or a very brief incubation at 37 degrees C). Under these restrictive conditions, latex binding sites were detected over the whole membrane whereas receptors for IgG-opsonized erythrocytes, aggregated IgG, complement-opsonized erythrocytes, and complement-opsonized zymosan were excluded from the protuberance. Thus, functional (endocytosis) and structural (inherent receptor distribution) analyses of membrane topography define different patterns of asymmetry in protuberant cells. The asymmetry induced in 1774.2 macrophages by colchicine is highly analogous to the functional and structural polarity of epithelial cells. Exploration of this analogy may provide insight into the development of polarized epithelia and, more generally, into mechanisms by which specialized areas of membrane are established.     

High throughput particle analysis: combining dielectrophoretic particle focussing with confocal optical detection

A micro flow cytometer has been fabricated that detects and counts fluorescent particles flowing through a microchannel at high speed based upon their fluorescence emission intensity. Dielectrophoresis is used to continuously focus particles within the flowing fluid stream into the centre of the device, which is 40 microm high and 250 microm wide. The method ensures that all the particles pass through an interrogation region approximately 5 microm in diameter, which is created by focusing a beam of light into a spot. The functioning of the device was demonstrated by detecting and counting fluorescent latex particles at a rate of up to 250 particles/s. A mixture of three different populations of latex particle was used, each sub-population with a distinct level of fluorescent intensity. The device was evaluated by comparison with a conventional fluorescent activated cell sorter (FACS) and numerical simulation demonstrated that for 6 microm beads, and for this design of chip the theoretical throughput is of the order of 1000 particles/s (corresponding to a particle velocity of 10 mm s(-1)).     

Interactions between protein-gold complexes and cell surfaces: a method for precise quantitation

We have developed a rapid and precise electron microscope technique for the quantitation of gold particles in suspension using latex microspheres as a reference (EM latex technique). This technique allowed us to determine the specific absorption of colloidal gold at its absorption maximum (520 nm) and the average number of ligands ([125I]IgG) bound to one gold particle. On the basis of these values important binding characteristics of protein-gold complexes to cell surfaces were analyzed in a model system consisting of Staphylococcus aureus with protein A on the cell wall as a specific binding site for IgG-Au. Our observations showed that the number of binding sites represented by one IgG-gold complex depended primarily on the particle size, with one 20-nm IgG-Au corresponding to 15 and one 6-nm IgG-Au to 2.5 binding sites. Hence, the efficiency of binding of IgG-Au complexes increased with decreasing gold particle size. Saturation of binding sites, however, was not achieved. The technique also made possible the determination of the affinity between IgG-Au complexes and the cell surface; this affinity can either be regarded as a characteristic of the ligand IgG or of the gold particle. We observed that the affinity of IgG decreased with the size of the gold particles to which IgG was bound, whereas the affinity of the entire gold particle increased with particle size. The EM latex technique for quantitation of gold particles extends the general use of protein-gold complexes to the quantitative characterization of their interaction with cell surface constituents.     

Development and application of thermo-sensitive immunomicrospheres for antibody purification

The latex particles composed of poly(styrene/N-isopropylacrylamide/glycidyl methacrylate) [P(St/NIPAM/GMA)] and poly(styrene/N-isopropylacrylamide/methacrylic acid) [P(St/NIPAM/MAA)] were prepared by emulsifier-free emulsion polymerization. These latex particles with submicrometer size showed the thermosensitivity originated from the thermo-sensitive nature of NIPAM. That is, the minimum NaCI concentration for flocculation of these latex particles [critical flocculation concentration (CFC)] decreased significantly with increasing temperature and reached constant values at above the critical temperature [critical flocculation temperature (CFT)]. At a certain NaCl concentration, the thermo-sensitive latex particles were flocculated by raising temperature, and conversely, the flocculated thermo-sensitive latex particles were completely dispersed by lowering temperature. Bovine serum albumin (BSA) was covalently immobilized onto the P(St/NIPAM/GMA) and P(St/NIPAM/MMA) latex particles with high efficiency. The BSA-immobilized P(St/NIPAM/GMA) and P(St/NIPAM/MAA) latex particles (immunomicrospheres) showed the similar dependencies of CFC on temperature to the bare latex particles. These thermo-sensitive immunomicrospheres were successfully used for the immunoaffinity purification of anti-BSA antibodies from antiserum. (c) 1994 John Wiley & Sons, Inc.     

Antibody-bearing liposomes improve agglutination of latex particles used in clinical diagnostic assays

Ligand-bearing liposomes are used to enhance the agglutination ‘signal’ of a typical latex assay for the detection of human rheumatoid factor. Heat-denatured IgG, the antigen to which rheumatoid factor binds naturally, was covalently attached to latex spheres. The liposomes were covalently coated with a ‘second ligand’ which also recognizes rheumatoid factor, anti-human IgM Fab′ fragments. In the present test configuration, rheumatoid factor present in a patient's serum binds to the IgG attached to the latex particles. The liposomes, in turn, bind rapidly to rheumatoid factor-sensitized latex, via the second ligand, promoting the formation of large, clearly visible latex aggregates. When latex spheres bearing the same type and density of second ligand were used to replace the liposomes they failed to improve agglutination, suggesting that multivalent presentation of the second ligand is not sufficient to insure the improvement. These results suggest that fluidity of the liposomal membrane is a requirement for the effect. Sensitivity as well as ‘readability’ are improved by the liposomes while specificity remains unaffected. The principle of using ligand-bearing liposomes to enhance particle agglutination is applicable to a wide range of other diagnostic assays.     

The preparation of an IgG diagnostic agent based on stained polyacrolein latexes for use in the latex agglutination reaction

IgG diagnosticum for measuring the concentration of 131I-labeled IgG antibodies to enteric antigen beta 1MA by the latex agglutination inhibition (LAI) test has been prepared on the basis of polyacrolein latexes. A method for the titration of anti-IgG antibodies with the use of the above diagnosticum has been developed, based on the late, agglutination (LA) test. The optimum conditions for the microtitration variant of the LA and LAI tests have been defined. High sensitivity, specificity and simplicity of analysis with the use of latex IgG diagnosticum have been demonstrated. The newly developed methods have been successfully used in laboratory trials of a new diagnostic radiopharmaceuticals for the assay of 131I-labeled antibodies in this preparation and for the detection of side effects of immunization on the recipients.     

Characterization of the interaction of human eosinophils and neutrophils with opsonized particles

The interaction of human eosinophils with opsonized particles was compared with that of human neutrophils. When eosinophils are stimulated with serum-opsonized zymosan particles, the lag time in H2O2 production is twice as long as found with neutrophils. Moreover, the concentration of these IgG + C3-coated particles required for optimal stimulation is about four times as high for eosinophils as for neutrophils. Under these conditions, the two cell types generate similar amounts of H2O2. However, eosinophils produce twice as much H2O2 as do neutrophils when stimulated with the soluble agent phorbol myristate acetate. Thus, although the oxidase capacity of eosinophils is larger than that of neutrophils, opsonized zymosan is a weak trigger for this activity in eosinophils. This phenomenon may be due to differences between the two cell types in the plasma membrane receptors or in the receptor oxidase transducing signal. The following are indications for the first possibility. i) IgG interacts poorly with the Fc gamma receptors on the eosinophil surface compared with those on neutrophils. This was shown by the inability of IgG-coated zymosan or IgG-coated latex to trigger any substantial H2O2 production by eosinophils unless brought into close contact with these cells by centrifugation. In contrast, neutrophils are stimulated by these particles both in suspension and in a pellet. The dissimilarity of the Fc gamma receptors on eosinophils and neutrophils was also shown with respect to antigenicity, determined by the monoclonal antibodies 3G8 and CLB-FcR-1. ii) Eosinophils contain about half as many receptors for C3b and C3bi on their surface as do neutrophils, also detected with monoclonal antibodies. The interaction of IgG subclasses with functional Fc gamma receptors on eosinophils and neutrophils showed that eosinophils release twice as much H2O2 as do neutrophils upon interaction with IgG1-, IgG2-, or IgG3-coated Sepharose beads, but this difference becomes fivefold with IgG4-coated Sepharose. This might be of relevance to the situation of chronic antigenic stimulation, e.g., in chronic schistosomiasis, in which eosinophil numbers and IgG4 antibody levels are elevated.