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Large liposome agglutination technique for the serological detection of syphilis.

In response to tissue invasion by Treponema pallidum, an antibody complex, called reagin, appears in the serum of the syphilitic patients. Reagin has the ability to combine with cardiolipin, therefore, cardiolipin has traditionally been used as the antigen in different test configurations for the serological detection of syphilis: the VDRL (Venereal Disease Research Laboratory) flocculation test and the RPR (rapid plasma reagin) test. Here we introduce a liposome preparation composed of cardiolipin, lecithin and cholesterol and containing a colored dye which upon mixing with syphilitic serum on a slide or a plastic card results in clearly visible liposome agglutination. We show that the liposomes in the size range of 1-10 micron are essential for proper sensitivity. The liposomes are colored with either a water-soluble dye entrapped in the aqueous space of liposomes or a lipophilic dye embedded in the membrane. By virtue of the liposome size and coloration the agglutination pattern is easily visible within 3-5 min without the use of a mechanical rotator. The approach described here can be extended to the detection of other antibodies and polyvalent antigens.

Agglutination Tests↗

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 micrograms/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.

Agglutination Tests↗

Field applications of agglutination and cytoadherence assays with Plasmodium falciparum from Papua New Guinea.

Plasmodium falciparum isolates obtained directly from patients in Papua New Guinea were tested in their first cycle of growth in vitro for adherence to melanoma cells and for susceptibility to agglutination by immune serum. Binding varied among isolates and, in many cases, increased with further rounds of replication under optimal culture conditions. Binding inhibition assays and agglutination assays demonstrated extreme heterogeneity of surface antigens; apparently none of the sera from adult patients recognized all of the variants presented.

Adult↗

Gelatin particle indirect agglutination test for mass examination for strongyloidiasis.

An indirect agglutination test using recently developed gelatin particles was assessed to determine its applicability as a screening test for mass examination for strongyloidiasis. 1199 individuals in Sashiki Town, Okinawa Island, were screened by the test and 34.7% were determined to be antibody positive. Follow-up examination of the persons whose sera showed positive antibody responses demonstrated the presence of faecal larvae in 41.7%. The calculated infection rate (14.5%) was similar (14.1%) to that indicated by another survey using the micro-enzyme-linked immunosorbent assay (micro-ELISA), conducted simultaneously among the inhabitants. The indirect agglutination test was simple to perform in a short time and without specialized equipment. Additionally, the gelatin particles have many advantages as an antigen carrier, e.g. in handling, reading of the resulting pattern, and stable, long-term preservation. The test was considered to be more convenient than the micro-ELISA for mass screening for strongyloidiasis.

Adult↗

Evaluation of an agglutination HIV-1 + 2 antibody assay.

Studies have shown that an HIV (HIV-PA) agglutination assay (Serodia) for the detection of antibody to human immunodeficiency virus (HIV) can be as sensitive and as specific as enzyme-linked immunosorbent assay (ELISA). However, since this HIV assay was designed to detect antibody to the HIV-1 virus, a substantial number of HIV-2 positive sera are missed by this assay. Since the HIV-2 has now been found throughout the world this test is becoming less suitable. The new HIV-1 + 2 assay version (HIV-1 + 2 PA) was evaluated in 300 sera, which contained 50 HIV-1, 40 HIV-2 and 10 HIV-1/HIV-2 antibody positive samples, and a sensitivity and specificity of 100% and 99%, respectively, was obtained. Whereas all HIV-2 positive sera were detected by the new HIV-1 + 2 version, 26% (13/50) were missed by the old version of the agglutination test. It is concluded that the HIV-1 + 2 PA assay is a promising instrument free assay which can be used for screening purposes in areas where both HIV-1 and HIV-2 are present.

AIDS Serodiagnosis↗

C-reactive protein in eel: purification and agglutinating activity.

C-reactive protein was highly purified from Japanese eel (Anguilla japonica) serum by precipitation with phosphatidyl-choline and Ca2+. On SDS-PAGE, eel C-reactive protein (eCRP) migrated as a single band with a molecular weight of 24,000 under reducing and 23,500 under non-reducing conditions. The molecular weight of native eCRP was estimated to be 120,000 by Sephacryl S-300 gel filtration. The eCRP was detected within the albumin region on immunoelectrophoresis. The eCRP showed an agglutinating activity against Streptococcus pneumoniae in the presence of Ca2+, and the activity was inhibited by 1 mM EDTA or 1 mM phosphorylcholine (PC). The eCRP also agglutinated rabbit red blood cells (RRBC), but not human and five other kinds of red blood cell. The hemagglutinating activity was inhibited by glucosamine or mannose. The eCRP formed a precipitin line with histone, protamine, poly(L-lysine) and poly(L-arginine) in agarose gel. The serum levels of eCRP were distributed in 6.8 ng/ml-5.3 mg/ml, n = 187, the mean value being 834 ng/ml.

Agglutination↗

Impaired agglutination of IgM resulting from non-enzymatic glycation in diabetes mellitus.

Non-enzymatic glycation of circulating immunoglobulin G, A and M, measured by boronic acid affinity chromatography was found to be significantly increased in diabetes mellitus. The mean value of glycated IgM was already high in the control group: 49.58% (+/- 10.3% SD) and increased up to a mean value of 61.00% (+/- 8.5%) in the diabetic group. Similarly IgG and IgA glycation was significantly higher in the diabetic group than in controls: IgG 21.6% (+/- 3.4%) vs. 14.1% (+/- 2.9%; P less than 0.01); IgA 14.7% (+/- 4.9%) vs. 7.7% (+/- 1.3%; P less than 0.01). To investigate how glycation would alter IgM function, serum proportions from diabetic patients with blood group O were separated into glycated and non-glycated fractions by affinity chromatography and, adjusted to the same concentrations, tested against group A1 erythrocytes. Agglutination, which is mainly an IgM-mediated reaction, was significantly lower in the glycated than in the non-glycated fraction of IgM. The correlation between glycation of IgM and the reduction of agglutination titres in the glycated fraction was significant (r = 0.88, P less than 0.001). We conclude that impaired IgM function may be caused by non-enzymatic glycation in diabetes mellitus with possible consequences for host resistance in the early phase of infection.

Agglutination↗

Agglutination assay of bladder cells by Concanavalin A proved the high susceptibility of analbuminemic rats to bladder carcinogens.

The susceptibility of analbuminemic rats, a mutant strain of Sprague-Dawley rats, to bladder carcinogens was examined by testing the agglutinability of isolated bladder epithelial cells by Concanavalin A (Con A). One-week treatment with 0.001% N-butyl-N-(4-hydroxybutyl)nitrosamine (BHBN) in the drinking water or 0.01% N-(4-(5-nitro-2-furyl)-2-thiazolyl)-formamide (FANFT), 0.01% and 0.1% 4-aminobiphenyl, 0.1% 4-nitrobiphenyl, 0.1% benzidine, 0.1% 2-napthylamine or 5% sodium saccharin in the diet clearly induced high agglutinability of bladder cells of male analbuminemic rats but not Sprague-Dawley rats. These results suggest that male analbuminemic rats are very susceptible to bladder carcinogens in general and therefore will be very useful in screening tests for bladder carcinogens.

2-Naphthylamine↗

Serological diagnosis of Trypanosoma evansi (Steel, 1885) in horses using a direct agglutination test.

A direct agglutination test is described to diagnose 'Mal de Caderas' caused by Trypanosoma evansi. The antigen used was a suspension of trypsin-treated parasites stabilized with formalin. The test was evaluated in horses with both natural and experimental infections. Test sensitivity and specificity were 94 and 97%, respectively. Treatment of serum with 2-mercaptoethanol before testing permitted the differentiation of IgM and IgG antibodies, and possible differentiation of current infection from past exposure to the parasite. The antigen was stable over a 6-month evaluation period and also showed good reproducibility between different batches. The direct agglutination test is proposed as another tool for diagnosis of T. evansi in horses, both for detecting clinical cases and for seroepidemiological studies.

Agglutination Tests↗

Comparison of a rapid latex agglutination assay and a fluorescent-antibody technique for the detection of herpes simplex antibody.

A total of 202 serum specimens was tested for the presence of herpes simplex virus antibody using a premarket latex agglutination kit (Wampole) and an indirect fluorescent antibody (IFA) technique (electronucleonics). Discrepant results between the two assays were resolved using an Enzyme-linked immunosorbent assay (ELISA) procedure. The overall sensitivity of the latex was 99.2% with a specificity of 98.5%. The latex agglutination test evaluated is a viable alternative to indirect immunofluorescence for the detection of herpes simplex virus antibody in serum samples.

Agglutination Tests↗

Vi-specific latex agglutination for early and rapid detection of Salmonella serotype typhi in blood cultures.

Latex particles coated with rabbit antisera against Salmonella serotype typhi (S. typhi) Vi and O (STO) antigens were used in slide agglutination tests for the rapid identification of S. typhi in blood culture broths as soon as Gram-negative bacilli (GNB) were detected in them. Among 231 consecutive blood cultures showing GNB tested for Vi, and a subset of 163 tested for STO, by latex agglutination (LA), 125 and 32, respectively, were positive. The GNB in 127 blood cultures were confirmed by conventional methods as S. typhi, 125 (98.4%) of which had been identified by the Vi LA test. In the subset of 163, 81 grew S. typhi, of which only 32 (39.5%) had been identified by the STO LA tests. Thus, the sensitivity of the Vi and STO LA tests was 98.4% and 39.5%, respectively, whereas the specificity was 100% for both tests. Of the S. typhi isolates, 38 (30.4%) were detected by the Vi LA test on day 2 and 73 (58.4%) on day 3, day 1 being the date of inoculation of the blood culture broths. Thus, the Vi LA test is suitable for the early and rapid confirmation of S. typhi in blood culture.

Agglutination Tests↗

Rapid serotyping of enteropathogenic Yersinia enterocolitica strains by co-agglutination.

A rapid co-agglutination test using monospecific antisera was developed for the serological typing of enteropathogenic strains of Yersinia enterocolitica. A total of 70 bacterial strains (17 reference strains and 53 clinical isolates) were examined. Absorption of immune sera against serotypes O:3, O:8 and O:9 with their heterologous antigens (S-LPS) was necessary to avoid the appearance of different cross-reactions, as observed by co-agglutination. The proteins present in the S-LPS preparations obtained from each serotype seemed to be responsible for such cross-reactions. Results obtained with a total of 57 clinical isolates belonging to other members of the family Enterobacteriaceae indicate a high specificity of the assay.

Agglutination Tests↗

Agglutination study of poly(allyl-alpha-D-glucopyranose/styrene) latex particles in the presence of Concanavalin A.

Latex agglutination of the glucose-modified latex, which were synthesized by emulsion copolymerization of allyl-modified glucose and styrene monomers by specific binding interactions between Concanavalin A (Con A) and allyl-alpha-d-glucopyranose, were investigated. The surface of the glucose-modified latex was characterized by dye-partition method and the number of glucose was 1517 per latex particle. The average particle size and the polydispersity index of the latex were 78.3 and 1.005, respectively. Time-evolution adsorption behavior of various concentrations of the Con A and the consequent latex agglutination were studied by UV spectrophotometer at 540 nm and zeta-potential analyzer at the fixed latex concentration of 0.02 wt%. Specific binding between Con A and allyl-alpha-D-glucopyranose leaded the latex particles to coagulate by decreasing the electrostatic repulsion between the particles and mobility.

Agglutination↗

Utility of an immunocapture-agglutination test and an enzyme-linked immunosorbent assay test against cytosolic proteins from Brucella melitensis B115 in the diagnosis and follow-up of human acute brucellosis.

The utility of an immunocapture-agglutination (Brucellacapt, Vircell SL, Granada, Spain) test and an enzyme-linked immunosorbent assay IgG, IgA, and IgM (ELISA-IgG, ELISA-IgA, ELISA-IgM) against cytosolic proteins from Brucella melitensis B115 (R) was compared with ELISA-IgG, ELISA-IgA, and ELISA-IgM against smooth lipopolysaccharide (S-LPS) from B. melitensis 16M (S), serum agglutination test (SAT), and Coombs test in the diagnosis and follow-up for 10 months of 51 patients with acute brucellosis. The sensitivities of ELISA tests against cytosolic proteins varied from 49.0 % for ELISA-IgG to 64.7% for ELISA-IgM and were lower than the sensitivities showed by ELISA S-LPS (from 88.2% to 92.2%), SAT (88.2%), Coombs (96.1%), and Brucellacapt (98.0%) tests. Specificity was over 93% in all cases. The evolutionary behavior of the SAT, Coombs, and Brucellacapt tests was similar. There was a decrease of between 20% and 40% in antibody titer in the 10th month of evolution after treatment. The evolutional curves of IgG, IgA, and IgM against cytosolic protein increased slightly till the eighth month. The specific IgM and IgA antibodies against protein fractions began to show a drop from the eighth month on, showing levels slightly lower than the initial sera values by the end of the 10th month. In this month, titers of specific IgG against proteins fractions remained higher than the titers showed by the initial sera.

Acute Disease↗

Rapid detection of Leishmania infantum infection in dogs: comparative study using an immunochromatographic dipstick rk39 test and direct agglutination.

A rapid, sensitive and specific tool for detection of Leishmania infantum infection in dogs, would be highly desirable, because it would allow control interventions in endemic areas of Zoonotic visceral leishmaniosis (ZVL). In this study, we compared an immunochromatographic dipstick test with direct agglutination test (DAT) for detecting L. infantum infections in dogs from areas of ZVL endemic in Iran. The validity of the dipstick rk39 (Cypress Diagnostic Company, Belgium) for canine visceral leishmaniosis (CVL) was compared with a standard direct agglutination test on 116 clinically suspected dogs and 152 healthy controls from endemic areas of Ardabil and East Azerbaijan provinces, north-western of Iran for 1 year. A sensitivity of 70.9% and specificity of 84.9% were found at a 1:320 cut off titer when DAT confirmed cases were compared with healthy control. As the dipstick rk39 test is rapid, noninvasive and does not require much expertise or elaborate equipment, it can be used for screening and diagnosis of canine visceral leishmaniosis in remote endemic areas.

Agglutination Tests↗

Procainized murine macrophages fail in the agglutination of opsonized isologous erythrocytes.

Murine peritoneal macrophages exhibit numerous slender microvilli and several invaginations of the cell membrane. By treatment with isotonic 2.10(-2) M procain-PBS the surface of macrophages is smoothed significantly. Isolated macrophages agglutinate opsonized isologous erythrocytes. Procainizing of macrophages inhibits this agglutination. The finding provides evidence for typing of macrophage receptors whether or not sensitive to procain treatment.

Agglutination↗

Immunoglobulins in the semen of men with azoospermia, oligospermia, or self-agglutination of spermatozoa.

One hundred samples of semen showing azoospermia, oligospermia, self-agglutination of spermatozoa, or normal physiologic spermatozoa were examined for evidence of autoimmune disorders. Immunoglobulin A, secretory immunoglobulin A, and complements C3 and C4 were present in a greater proportion of abnormal samples than in normal ones. The immunoglobulin A content of the abnormal samples was markedly increased, and the immunoglobulin G level slightly increased, as compared with normal samples. These findings support the hypothesis that agglutination of spermatozoa in semen is an autoimmune mechanism. They also suggest that, although spermatozoa may activate the mechanism, it is possible that abnormalities of the male genital tract can stimulate the formation of spermatozoal antibodies.

Complement C3↗