The Latex Serologic Test for Syphilis—A New Screening Procedure

A new macroscopic screening test for syphilis, the Latex-sts test, is extraordinarily simple. After inactivation of the patient''s serum for 30 minutes at 56°C the test is performed by mixing the patient''s serum with latex particles coated with cardiolipin and a protein fraction obtained from the non-pathogenic Reiter strain of Treponema pallidum. Two to three minutes after mixing, the result of the test is observed on a ringed serologic plate. The sensitivity, specificity and reproducibility of the new test are equivalent to those of the qualitative Venereal Disease Research Laboratory tube test. The advantages of the Latex-sts are that it can be done in a short time, it is simple and it requires a minimum of laboratory equipment. The coated latex particles are stable for 12 months.     

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).     

A rabies agglutination test (RAT) for rabies antibody detection

An agglutination test has been developed for the detection of rabies antibodies after human vaccination. The rabies agglutination test (RAT) is based on the capability of specific antibody to agglutinate sensitized polystyrene (or latex) beads. In the RAT, latex beads were coated, in a first step, with inactivated and purified rabies virus (PV strain adapted and propagated on BHK-21 cells) and, in a second step, with bovine serum albumin. Negative control beads were coated with bovine serum albumin (BSA) only. To test for human antibody, several microliters of serum were mixed on a glass slide with an equal volume of virus-sensitized beads and the mixture was gently agitated. After a few minutes, agglutination was visible with sera which had been characterized as positive by the virus neutralization antibody (VNAb) technique. No agglutination was observed with negative sera tested with virus-coated beads or with positive sera tested with BSA-coated beads. Virus-sensitized beads were agglutinated when the virus neutralizing antibody titres were equal to or greater than 2.5 international units per ml (IU/ml) in human sera. The concordance between the RAT results and VNAb titres was about 97% when 2.5 IU/ml was taken as the cut off value for determining the positive sera with the VNAb technique. The possibility that clinicians might use the RAT as a simple means to determine sero-conversion at the end of the post-exposure treatment of patients is discussed.     

Large-scale validation of a latex agglutination test for diagnosis of tuberculosis

Large-scale validation of a simple latex agglutination test for the diagnosis of tuberculosis is described. Soluble antigens extracted from a non-pathogenic saprophytic mycobacterium, Mycobacterium w, which shares antigenic determinants with Mycobacterium tuberculosis, were covalently linked to carboxylated polystyrene latex beads. Batch to batch reproducibility of coated latex was ensured. Latex reagents were standardized to overcome non-specific agglutination. Reagents of the test are stable for 1 year at 4 degrees C. A total of 1,058 serum samples of pulmonary and extrapulmonary tuberculosis patients or patients with other pulmonary diseases and healthy controls living in endemic areas were tested. Sensitivity of 94% for pulmonary tuberculosis and 87% for extrapulmonary tuberculosis was obtained. Specificity is 92.2% for healthy controls and patients with other respiratory diseases. We conclude that the latex agglutination test can be utilized for mass screening for both pulmonary and extrapulmonary tuberculosis where diagnosis by existing methods is much more difficult.     

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.     

Assessment of three simple techniques for the screening of circulating immune complexes: Correlation with a parameter of complement consumption

The sera of 36 normal controls, 45 patients with various diseases and 11 pregnant women were screened for circulating immune complexes using three relatively simple and inexpensive techniques. These included inhibition of agglutination of IgG coated latex particles with a serum having rheumatoid factor activity, polyethylene glycol precipitation and anti-complementary activity test. The circulating immune complexes were detected in a significantly higher proportion of patients as compared to normal controls. In the patients, the presence of circulating immune complexes did not always correlate with clinically detectable immunoinflammatory tissue damage indicating that pathogenic as well as nonpathogenic immune complexes were being detected by the above mentioned techniques. The alpha-1-antitrypsin/C3 ratio, however, correlated well with clinically apparent immuno-inflammation.     

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.     

A simple, rapid, and sensitive method of determining CSF IgG, using latex

A simple, rapid and sensitive method of determining cerebrospinal fluid IgG is presented. The procedure depends upon the fact that spinal fluid gamma globulin, and two of its components (IgG and IgM) will precipitate latex particles in proportion to their concentration, and that the optical density of the supernatant solution containing unprecipitated latex particles after centrifugation is inversely proportional to the concentration of these immunoglobulins. The method is sensitive to 50 nanograms, is inexpensive, requires 0.1 ml. or less of spinal fluid, and can be performed in minutes. Preliminary studies show that it compares favorably with results obtained by radial immunodiffusion.     

The effect of 2-deoxyglucose on guinea pig polymorphonuclear leukocyte phagocytosis

The effects of 2-deoxyglucose (DOG), an inhibitor of glycolysis, on guinea pig polymorphonuclear leukocytes (PMN) obtained from peritoneal exudates was examined. ATP levels in PMN were reduced by 40% by one hour following an incubation with 2-deoxyglucose. When complement (C3) coated ¹⁴C-staphylococcus aureus, C3 coated lipopolysaccharide-paraffin oil droplets (LPSPO), ¹⁴C-pneumococcus opsonized with IgG, or albumin coated paraffin oil droplets opsonized with IgG were added to cell suspensions containing DOG, the phagocytizing rate was 1,310 ± 55 cpm/5 x 10⁶ cells/15 minutes, 6 ± 2 μg paraffin oil (PO)/10⁷ cells/minute, 2,250 ± 175 cpm/1 x 10⁶ cells/20 minutes or 0.037 ± 0.01 mg PO/10⁷ cells/minute compared to control values of 5,970 ± 275 cpm/5 x 10⁶ cells/15 minutes, 35 ± μg PO/10⁷ cells/15 minutes, 4,510 ± 200 cpm/1 x 10⁶ cells/20 minutes and 0.067 ± 0.01 mg PO/10⁷ cells/minute. In parallel studies the phagocytic index for latex was 0.74 ± 0.28 in DOG compared to control of 2.36 ± 1.13 and the phagocytic rate of albumin coated paraffin oil droplets was 0.029 ± 0.01 mg PO/10⁷ PMN/minute in DOG compared to control of 0.048 mg PO/10⁷ cells/minute. When ATP levels were maintained by the simultaneous addition of 5 mM glucose or pyruvate to media containing DOG, latex ingestion was improved to 1.15 ± 0.3 with glucose and 1.59 ± 0.64 with pyruvate and albumin coated particles to 0.045 ± 0.01 mg PO/10⁷ PMN/minute with pyruvate. There was no improvement in the uptake of either the C3 dependent particles or IgG coated Pneumococci in media containing DOG and glucose and/or pyruvate. Following the removal of DOG from the extracellular medium and the addition of pyruvate or glucose, phagocytosis of C3 dependent LPS-PO was restored to normal values. Neither the binding of C3 or IgG coated particles to the PMN nor the lateral movement of glycoprotein utilizing concanavalin A capping was affected by DOG. Thus, the presence of DOG in the PMN containing adequate amounts of ATP will selectively and reversibly inhibit those surface events required for phagocytosis of C3 and IgG bound particles but not latex particles or albumin particles which non-specifically bind to PMN.     

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.     

Use of heterophilic mononucleosis antigen in the latex diagnostic test. III. Diagnostic field studies

The aim of this study was to evaluate usefulness of latex coated with a preparation of heterophilic mononucleosis antigen for the detection of antibodies present during a course of infectious mononucleosis. Studies were performed in district serological laboratories. Sanitary-Epidemiological Stations in conditions of routine diagnostics. Studies were performed on 656 blood serum samples collected from individuals with a clinical suspicion of infectious mononucleosis. Latex and Paul-Bunnell-Davidsohn (PBD) tests were run in parallel. Out of 154 blood serum samples which contained antibodies detected by PBD test in diagnostically significant titer of 1:56 or higher, 151 were reactive in latex test in the dilution 1:5 or higher, while in the remaining three cases agglutination appeared with undiluted serum only. Out of 268 serum samples tested in latex test 167 reacted in 1:5 titer - diagnostically accepted as a significant or in higher titers. The sensitivity of latex test amounted to 98.1% and specificity was 96.9% as compared to reference Paul-Bunnell-Davidsohn test. The results of our study suggest the possibility of replacing Paul-Bunnell-Davidsohn test by latex test performed as a method providing the possibility of determination of the presence of heterohilic antibodies in blood serum samples and the follow up of their dynamics.     

Scanning electron microscopy of in vitro serum-mediated destruction of juvenile Fasciola hepatica

The tegumental surface of immature Fasciola hepatica was damaged when incubated in vitro with serum collected from an experimentally infected calf. Degeneration of the tegumental surface was observed by scanning electron microscopy (SEM) at 4 hr. after incubation. Decomposition was observed 8 to 12 hr after incubation and complete destruction of the tegument occurred by 16 hr. The flukes became inactive after 8 to 12 hr of incubation. None of the above findings were observed for the tegument of flukes incubated in tissue culture media or in media containing normal calf serum and the trematodes remained motile throughout the incubation period. Latex particles were used as an immunological marker for SEM studies to determine if gamma globulin could be responsible for the observed changes and, if so, the site of antibody attachment. The coated latex particles covered the entire surface of flukes recovered from mice 5 days after infection with metacercariae. In contrast, latex particles coated with either normal gamma globulin or gamma globulin from serum of the experimentally infected calf that had been adsorbed with disrupted adult flukes were not attached to the surface of the flukes. Absorption of the serum with disrupted, adult flukes decreased the concentrations of immunoglobulins (Ig)G1 and G2 whereas IgA and IgM were apparently not affected.     

Applications of ultrasound streaming and radiation force in biosensors

Direct radiation force (DRF) and acoustic streaming provide the main influences on the behaviour of particles in aqueous suspension in an ultrasound standing wave (USW). The direct radiation force, which drives suspended particles towards and concentrates them in acoustic pressure node planes, has been applied to rapidly transfer cells in small scale analytical separators. The DRF also significantly increased the sensitivity of latex agglutination test (LAT) by concentrating the particles of an analytical sample in the pressure node positions and hence greatly increasing the antibody-antigen encounter rate. Capture of biotinylated particles and spores on a coated acoustic reflector in a quarter wavelength USW resonator was DRF-enhanced by 70- and 100-fold, respectively compared to the situation in the absence of ultrasound. Acoustic streaming has been successfully employed for mixing small analytical samples. Cavitation micro-streaming substantially enhanced, through mixing, DNA hybridization and the capture efficiency of Escherichia coli K12 on the surface of immunomagnetic beads. Acoustic streaming induced in longitudinal standing wave and flexural plate wave immuno-sensors increased the detection of antigens by a factor of five and three times, respectively. Combined DRF and acoustic streaming effects enhanced the rate of the reaction between suspended mixture of cells and retroviruses. The examples of a biochip and an ultrasonic immuno-sensor implementing the DRF and acoustic streaming effects are also described in the review.     

Human monocyte spreading in vitro--inducers and effects on Fc and C3 receptors.

Human peripheral blood monocytes undergo cytoplasmic spreading following attachment to a glass surface. The extent of spreading is greater in the presence of antigen-antibody complexes, manganese, subtilisin and dithiothreitol. The human blood monocyte spreads more rapidly than the mouse peritoneal macrophage and is not inhibited by serum. Fc receptor activity is diminished when spreading is induced by antigen-antibody complexes and is not affected by other inducers. The binding of erythrocytes coated with C3 and the ingestion of latex particles are not inhibited during cytoplasmic spreading.     

Serological Activity of Staphylococcal Polysaccharide

The polysaccharide from cell walls of coagulase-positive staphylococci coated both latex particles and tanned red cells for agglutination by human sera and by specific staphylococcal antisera. Treatment with trypsin or autoclaving destroyed the capacity of polysaccharide to coat particles but did not affect precipitation of antibody. Periodic acid destroyed both properties. The teichoic acid portion of the staphylococcal polysaccharide displayed precipitin activity similar to polysaccharide, but it did not coat either latex particles or tanned red cells. Teichoic acid did, however, inhibit specific agglutination of polysaccharide-coated particles or cells.     

Heterophilic infectious mononucleosis antigen used in the latex diagnostic test. II. Preliminary evaluation of the usefulness of latex reagents for detection of serum heterophile antibodies in patients with infectious mononucleosis

The aim of this study was to evaluate the usefulness of laboratory made reagent of polystyrene latex coated with three preparations of mononucleosis antigen (reagent MZ-I, MZ-II, and MZ-III) for detection of heterophile antibodies in patients sera with clinical symptoms of infectious mononucleosis. These studies were carried out on 153 serum samples taken from persons suspected of being infected with EB virus and on 100 healthy controls--blood donors. The results of latex tests were compared to the results of Paul-Bunnell-Davidsohn (PBD) and hemolytic tests. Out of three latex reagents evaluated only one (reagent MZ-I) showed sensitivity equal to PBD and haemolytic tests. The sensitivity reached 91.1%. Agglutination reaction when appeared in latex test at serum dilution 1:5, is considered positive and diagnostically significant result.     

Fibronectin-coated beads are endocytosed by cells and align with microfilament bundles

Carboxylated latex beads coated with fibronectin bind to the surfaces of NIL8 hamster cells. Similar beads coated with bovine serum albumin, gelatin or normal rabbit immunoglobulin do not bind to the cells, whereas beads coated with polylysine or rabbit anti-hamster immunoglobulin bind, as do fibronectin-coated beads. Surface-bound beads are endocytosed by the cells. This endocytosis is inhibited by N-ethyl maleimide or low temperatures. The endocytosed beads form arrays aligned with the microfilament bundles inside the cells and after extended incubations (18–24 h) become concentrated in the perinuclear region. These results suggest that, after phagocytosis of large particles, the endocytic vesicles align with microfilament bundles, as previously reported for coated pits involved in receptor-mediated endocytosis. The system described here offers possibilities for the analysis of membrane and transmembrane interactions involved in cell-substratum binding and phagocytosis.     

<Emphasis Type="Italic">Herpesvirus ateles</Emphasis>, a New Lymphoma Virus of Monkeys

THERE has been a marked increase of post-transfusion hepatitis associated with the increased utilization of blood and its products. The discovery of a reaction of serum from multiple transfused haemophiliacs with serum from patients suffering from hepatitis^1,2 has made it possible to analyse the hepatitis associated antigen (HAA), the agent associated with the transmittance of hepatitis. HAA is currently detected using haemophiliac serum as the antibody source and standard serological techniques, that is, immunodiffusion, complement fixation and immunoelectro-osmophoresis³. This report describes a latex agglutination procedure which is compatible with the existing technology in blood banks as well as being very rapid and sensitive. The assay is based on the agglutination of antibody-absorbed latex particles by HAA contained in the test serum.     

Intracellular delivery of nanometric DNA particles via the folate receptor

The size of condensed DNA particles is a key determinant for both diffusion to target cells in vivo and intracellular trafficking. The smallest complexes are obtained when each DNA molecule collapses individually. This was achieved using a designed cationic thiol-detergent, tetradecyl-cysteinyl-ornithine (C(14)COrn). The resulting particles were subsequently stabilized by air-induced dimerization of the detergent into a disulfide lipid on the DNA template. Particles are anionic (zeta potential = -45 mV), and their size (30 nm) corresponds to the volume of a single plasmid DNA molecule. The electrophoretic mobility of the condensed DNA, though quasi-neutralized, was found higher than that of the extended DNA. Moreover, the dimerized (C(14)COrn)(2) lipid was found to be an efficient transfection reagent for various cell lines. In an attempt to achieve extended circulation times and to target tumors by systemic delivery, we have coated the particles with PEG-folate residues. Plasmid DNA was condensed into monomolecular particles as described above and coated by simple mixing with DPPE-PEG-folate. Physicochemical measurements showed particles coated with 2% of DPPE-PEG(3400)-folate remain monomolecular and are stable in the cell-culture medium. Caveolae-mediated cell entry was demonstrated by ligand-dependence, by competition with excess folic acid as well as by confocal microscopy.