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PubMed1981

A sol particle agglutination assay for human chorionic gonadotrophin.

Conjugates, prepared by adsorption of antibodies on colloidal gold particles, were used in a homogeneous sol particle immunoassay (SPIA) for human chorionic gonadotrophin (HCG). The technique is based on sol particle agglutination, resulting in colour reduction. Optimal results were obtained using buffered conjugates prepared from 50 nm particles. Addition of polyethylene glycol (PEG) 6 000 to the conjugates, up to 15 g/l, increased the agglutination rate considerably. The optimal PEG 6 000 concentration of the conjugate depended on the desired incubation time and measuring range. The influence of temperature on the agglutination was negligible in the temperature range between 4 and 45 degrees C. Higher conjugate concentrations resulted in steeper dose-response curves. However, the measuring range (between 62.5 and 2 000 IU/l HCG) and the detection limit (approx. 50 IU/l HCG) were about the same. The dose-response curves for HCG dissolved in buffer or in urine were almost identical and their reproducibility was satisfactory. In our experience, homogeneous SPIAs have a high practicability, are easy to automate and provide an interesting new tool for the measurement of a variety of analytes.

Absorption↗

PubMed1990

Immunological detection of mycobacterial antigens in infected fluids, cells and tissues by latex agglutination. Animal model and clinical application.

We devised an immunoassay for the detection of mycobacterial antigens in cell lysates and in tissue extracts which is based on the agglutination of latex particles coated with anti-Mycobacterium bovis F(ab')2, followed by counting of non-agglutinated particles. Mycobacterium bovis cell lysates were tested and a reference curve was established, having a lower limit of detection of 15-20 Mycobacteria. We were able to detect mycobacterial antigens in cell lysates from bronchoalveolar washings and in spleen and liver lysates obtained from experimentally infected rabbits. Antigens were also detected in ten out of 11 samples obtained from patients with proven tuberculous infection. These samples were readily distinguished from 32 negative control samples after pepsin treatment. In contrast, periodate treatment of samples to destroy carbohydrate, abolished all reactivity. Following gel filtration chromatography we identified three peaks with antigenic properties in samples of all types. The detection of mycobacterial carbohydrate antigens by latex agglutination and particle counting should be a useful adjunct in the diagnosis of tuberculosis.

Agglutination Tests↗

PubMed1976

A new agglutinating activity from wheat flour inhibited by tryptophan.

A new compound endowed with agglutinating activity, designated the flour agglutinin, was extracted from wheat flour with water and purified by gel filtration and ion-exchange chromatography. The haptenic inhibitors of the plant agglutinins do not affect flour agglutinin activity which, on the other hand, is inhibited by D- and L-tryptophan. Flour agglutinin has a molecular weight of about 5 - 10(4) as determined by gel filtration. It consists of a neutral heteropolysaccharide constituted of D-xylose and L-arabinose, and is homogeneous as judged by sedimentation analysis. Flour agglutinin activity is destroyed by treatment with Cellulase 2000 and periodate, but is not affected by alpha-amylase and proteolytic enzymes. Compared to germ agglutinin, flour agglutinin exhibits a peculiar range of cell specificity. It agglutinates several normal cell types, but has no effects on some neoplastic cells tested. Tryptic digestion of erythrocytes does not affect their susceptibility to flour agglutinin-induced agglutination.

Agglutination Tests↗

PubMed1977

Cell surface microvilli and cell agglutinability.

Detached cells of some transformed mouse fibroblast lines have a villous surface whereas similarly treated cells of other lines are relatively smooth. These differences in surface morphology of detached cells are not reflected in their agglutinability with ConA and they cannot unambigously be explained from their morphology in situ. Treatments of normal and transformed Swiss mouse fibroblasts that induce marked changes in agglutinability with ConA do not cause equivalent changes in surface morphology. It is, therefore, unlikely that agglutinability of mouse fibroblasts by ConA is determined by the number of microvilli on the cell surface.

Agglutination↗

PubMed1992

Agglutination of Bordetella species by lectins.

Phase I cells of Bordetella pertussis but not those of B. parapertussis, B. bronchiseptica or B. avium were agglutinated by Limulus polyphemus lectin. Most strains of B. pertussis but not those of the other species were also agglutinated by Helix pomatia lectin. In precipitation reactions between lectins and purified Bordetella lipopolysaccharide (LPS) preparations a similar pattern occurred. Lectin agglutination provides a rapid presumptive method for the differentiation of B. pertussis from B. parapertussis and other Bordetella species.

Agglutination Tests↗

PubMed1987

Induction of lymphocyte agglutination and lysis by Newcastle disease virus.

Five strains of Newcastle disease virus (NDV) were tested for their ability to agglutinate and lyse chicken lymphocytes. All 5 strains agglutinated lymphocytes, and the agglutination was inhibited by anti-NDV sera. All 5 strains also caused a different degree of direct virus-induced lysis of 51Cr-labeled chicken lymphocytes. This direct lymphocyte cytotoxicity was lost after heating at 56 degrees C for 30 min, or after treatment with a high concentration of formalin (1:100 dilution). Neither a low concentration of formalin (1:5000) nor trypsin treatment had any effect on cytotoxicity.

Agglutination↗

PubMed1995

Agglutination of chicken lymphocytes by infectious bursal disease virus.

Four serotype 1 field strains of infectious bursal disease virus (IBDV) and one reference standard serotype 1 strain were tested for their ability to agglutinate peripheral blood lymphocytes of chickens. All the five strains agglutinated chicken lymphocytes. The agglutination was not inhibited by serotype 1 reference anti-IBDV serum. These results suggest the need for detailed analysis of IBDV ligand(s) and cell surface receptor(s) relationships.

Agglutination↗

PubMed2004

Detection of PCR amplicons from bacterial pathogens using microsphere agglutination.

For rapid and inexpensive detection of polymerase chain reaction (PCR) amplicons, a novel microsphere agglutination assay has been developed. PCR is carried out using biotinylated forward and reverse primers, and the amplified DNA fragments are able to agglutinate streptavidin-coated microspheres (5.7 microm in diameter). Purification of PCR amplicons is unnecessary when initial primer concentrations are 250 nM. Agglutination can be identified visually within 2 min without any additional equipment or reagents. Using listeriolysin (lisA)-specific biotinylated primers, we have successfully detected and identified Listeria monocytogenes lisA+ cells among Salmonella typhimurium, Staphylococcus aureus, Campylobacter jejuni and Escherichia coli O157:H7 cells. The simplicity of this protocol considerably reduces the time and cost of diagnostic PCR experiments. This procedure is potentially useful for various studies and field applications.

Agglutination↗

PubMed2006

A new in-vitro agglutination technique for potency estimation of antisnake venom serum (ASVS).

Traditionally the potency of ASVS is assayed quantitatively by in-vivo neutralization test for lethality in mice. A sensitive and simple in-vitro agglutination assay for the quantitative determination of Antisnake Venom Serum (ASVS) potency is reported. The method is rapid, cheap, simple, economical and above all does not require the use of experimental animals for potency assay of in process, unpurified and purified sera batches. Among in-vitro procedures, agglutination assay was favored in comparison to flocculation as the later was found to give variable results and also time consuming (high Kf value). Before application, the method was standardized and validated for choice and concentration of particulate material (latex vs. bentonite), temperature and optimum antiserum concentration. It is well known fact that venoms lose toxicity on dilution however this study demonstrated that the bentonite adsorbed venoms of the entire four snake species viz., Cobra, Krait, Russell's viper and Echis are stable even up to 30 days of storage. Among five lots each of unpurified serum, unprocessed plasma and purified sera tested, the results were found comparable with universally accepted in-vivo biological assay. The coefficient of correlation was found to be near 1.0 within 95% fiducial limits of acceptance and also significantly less variation was observed in the mean potency values and standard deviations. For all results p value was observed to be <0.01. Results indicate that in-vitro agglutination assay is suitable and can be used for potency estimation of in process as well as unpurified and purified ASVS batches.

Adsorption↗

PubMed2002

Sero-epidemiological assessment and diagnosis of visceral leishmaniasis in an endemic locality using Fast Agglutination Screening Test (FAST).

The Fast Agglutination Screening Test (FAST) was employed on sera obtained from an endemic area of visceral leishmaniasis in southwestern Ethiopia, in February 2000. The study involved (i) active case detection among 1575 residents of two villages; and (ii) passive case detection in an outpatient clinic. Sera of 1587 individuals, including 143 sera of previously treated VL patients, were tested. Based on the size of agglutination mat, the FAST results were read qualitatively as non-reactive (-), weakly reactive (1+), moderately reactive (2+) and highly reactive (3+). All FAST reactive sera were re-tested with the Direct Agglutination Test (DAT). After clinical screening of 1625 individuals, 61 individuals with signs and symptoms of early or late VL were found; 26 sera were FAST positive. Twenty-two of these suspected VL cases were subjected to parasitological examination using lymph node aspirates. Eighteen (81.8%) were confirmed either by demonstration of amastigotes in smears or promastigotes in NNN cultures. FAST reactive anti-leishmanial antibodies were detected in 4.5% of untreated and 70.6% of previously treated patients. Forty-five sera of 1390 previously untreated asymptomatic individuals (3.2%) were found to be FAST positive. This report demonstrates that FAST is a rapid and cost-effective screening test for the diagnosis and sero-epidemiological surveillance of visceral leishmaniasis.

Adolescent↗

PubMed2002

Detection of anti-ganglioside antibodies in Guillain-Barré syndrome and its variants by the agglutination assay.

Sera from 40 patients with Guillain-Barré syndrome (GBS), including the subtypes acute inflammatory demyelinating polyneuropathy (AIDP), acute motor axonal neuropathy (AMAN), acute motor and sensory axonal neuropathy (AMSAN), and Miller Fisher syndrome (MFS) were examined for the presence of anti-ganglioside antibodies using the ganglioside agglutination assay, and the enzyme-linked immunosorbent assay (ELISA). In the ELISA system, sera were tested for IgM and IgG antibodies to GM1, GM2, GD1a, GD1b, GT1b, and GQ1b gangliosides. Antibodies to gangliosides were detected in 21 (53%) of the GBS patients by agglutination assay and in 17 (43%) of the patients by ELISA. Some of the sera reacted with more than one ganglioside. Antibodies were not found in the control sera that were studied. The agglutination assay may be useful for rapid screening of GBS sera for antibodies to multiple gangliosides.

Agglutination Tests↗

PubMed1999

Sperm-lectin agglutination combined with swim-up leads to an efficient selection of highly motile, viable and heterogeneous ram spermatozoa.

Lectins, high molecular weight glycoproteins with different sugar-binding specificity, are able to agglutinate different cell types. The recovery of high-quality spermatozoa can be facilitated by the agglutination induced by the lectin binding. The objective of this study was to combine sperm-lectin agglutination with a dextran/swim-up procedure for developing a new selection technique for ram spermatozoa. To study sperm quality, cell viability (plasma membrane integrity), the HOS-test response and progressive individual motility were assessed. Simultaneously, centrifugal countercurrent distribution (CCCD) in an aqueous two-phase system was carried out to analyze sperm surface heterogeneity. Semen from 3 mature Saltz rams was pooled, and 0.5-mL aliquots were incubated with 4 fluorescein-labelled lectins (ECL, JAC, PSA, RCA). Then, a dextran solution was gently added and overlaid with medium. The top layer of the medium containing the spermatozoa was collected and replaced by careful addition of fresh medium. The incubation sequence was repeated 3 times at 10-min intervals. The consecutive 4 top layers obtained were pooled to give the swim-up combined sample. The highest rate of improvement in sperm quality was obtained after incubation with RCA, with a 50% increase in progressive individual motility, 21.6% in HOS value and 39.5% in viability. Total cell recovery was 64% (1.56x10(9) cells), with a viable cell recovery rate of 86%. The obtained sample showed 82% motility, 80% HOS score and 77% viability, up from the pre-swim-up values of 51, 60 and 57 %, respectively. Comparative CCCD analysis revealed a very high heterogeneous population in the RCA/swim-up sample obtained, while a much more homogeneous population was obtained in the sample after the dextran/swim-up procedure previously developed byus With this simple method, a large proportion of highly-motile spermatozoa with preserved plasma membrane and high heterogeneity can be obtained. These results strongly suggest that this selection procedure could result in a high fertility rate.

Animals↗

PubMed1999

Standardisation of subjectively scored HIV immunoassays: developing a quality assurance program to assist in reproducible interpretation of results using an anti-HIV particle agglutination assay as a model.

Immunoassays such as particle agglutination assays, rapid tests and western or line blots are scored or read subjectively. These readings display intra- and inter-reader variability, as well as intra- and inter-laboratory variability. In the present study the consistency of scoring was assessed between readers both within and between two groups of scientists using the Serodia anti-HIV particle agglutination assay as an example of an assay scored subjectively. An anti-HIV positive sample in eight serial dilutions made to yield a full range of results expected for the assay was presented 12 times (96 test wells). Each dilution was placed randomly in a plate and tested with the Serodia anti-HIV particle agglutination assay then photographed. Participants in the two groups each scored the photographed plate independently and twice, 2 h apart. Each well was assigned a status (the consensus result of the four most experienced Australian readers) and each participant's results were compared with this status. The average percentage of wells assessed as 'correct' for the Group A participants was 86% (range 56-98%) and for the Group B participants was 67% 'correct' (range 46-88%). In general, strongly positive and negative wells were scored 'correctly'. The highest variations between scores were seen in the borderline positive dilutions +/- region. A quality assessment program based on the method used to obtain these results will be instituted in order to improve the consistency of scoring assays read subjectively.

AIDS Serodiagnosis↗

PubMed2002

Development of a microslide agglutination assay with the aid of an inexpensive projection microscope.

A microslide agglutination assay was developed involving the mixing of 2.5 microl each of antiserum and a cell suspension of Listeria monocytogenes. Cell agglutination in the final volume of 5.0 microl was visually observed at a direct magnification of 22 x on the projection screen of an inexpensive 20 US dollar projection microscope. The procedure has the advantage of increasing by a factor of 20 the number of agglutination assays that can be performed with a given volume of antiserum with the use of an inexpensive optical projection system.

Agglutination Tests↗

PubMed1999

Ultrasonic enhancement of coated particle agglutination immunoassays: influence of particle density and compressibility.

The detection rate and sensitivity (analyte concentration limit) of coated particle agglutination immunoassays are increased in ultrasonic standing waves. The influence of particle volume, density and compressibility, properties that modify the ultrasonic radiation, and interaction forces the particles experience, on assay sensitivity with latex and silica particles in the range 0.25-1.0 microm is examined here. Streptavidin-coated 0.3-microm silica particles and 0.25-microm and 1.0-microm latex particles were examined for agglutination with biotinylated bovine serum albumin (bBSA) following exposure on axis in a 4.6-MHz radial standing wave. The lowest detection limit, 2 ng/mL bBSA, was achieved with the 0.3-microm silica. The detection limit decreased with increasing latex particle size. The limit of an ultrasound-enhanced agglutination immunoassay of rabbit antimouse immunoglobulin was 6-fold better with 1.0-microm coated silica than with equal-sized latex particles. Calculations show that the particle density-dependent ultrasonic interaction force dominates the particle compressibility force for the present case. The density-dependent force on silica, but not on latex particles, is shown to be comparable in magnitude to both the long-range van der Waal's attractive force and the electrostatic repulsion between the particles. This density-dependent force may explain the improved enhancement of analyte detection by coated silica compared with latex particles.

Agglutination Tests↗

PubMed1985

Structure-activity relationships in the dodecapeptide alpha-factor of Saccharomyces cerevisiae: position 6 analogues are poor inducers of agglutinability.

Five des-Trp1,Cha3,X6 analogues of alpha-factor, where X = Ala, Val, Ile, Nle, or D-Leu and X = Leu in the natural alpha-factor sequence, were prepared by solution-phase techniques utilizing isobutyl chloroformate or 1-hydroxybenzotriazole accelerated active esters as the coupling agents. Purification to 98% or greater homogeneity was accomplished by high-performance liquid chromatography on a reversed-phase muBondapak C18 column with methanol/water/trifluoroacetic acid as the mobile phase. Three of the synthesized analogues (X6 = Val, Ile, Nle) induced morphogenesis and increased agglutinability in a cells. These substitutions demonstrate that a gamma-branched side chain at position 6 is not essential for biological activity. All of the active analogues induced morphogenesis at lower concentrations than they induced enhanced agglutinability. These results and other structure-activity relationships [Baffi, R. A., Shenbagamurthi, P., Terrance, K., Becker, J. M., Naider, F., & Lipke, P. (1984) J. Bacteriol. 158, 1152-1156] indicate that the agglutination and morphological responses to alpha-factor can be varied independently. Replacement of Leu6 with Ala or D-Leu resulted in inactive analogues that were not antagonistic for alpha-factor activity. Cell-mediated hydrolysis experiments indicated that the biological activities of the alpha-factor analogues are independent of their rates of degradation. All position 6 analogues were hydrolyzed more slowly than the parent compound, suggesting that the enzyme which degrades alpha-factor is highly specific for the native structure.

Agglutination↗

PubMed1999

Agglutinating activity of alcohol-soluble proteins from quinoa seed flour in celiac disease.

The edible seeds of the quinoa plant contain small quantities of alcohol-soluble protein which, after peptic-tryptic digestion, are unable to agglutinate K562(s) cells. When separated by affinity chromatography on sepharose-6B coupled with mannan, peptic-tryptic digest separated in two fractions. Fraction B peptides (about 1% of total protein) were shown to agglutinate K562(s) cells at a very low concentration, whereas peptides in fraction A and in the mixed fraction A+B were inactive, suggesting that fraction A contains protective peptides that interfere with the agglutinating activity of toxic peptides in fraction B.

Agglutination↗

PubMed1977

Detection of sensitized human blood lymphocytes by agglutination with basic peptides: a possible test for malignant disease.

A simple rapid micro-agglutination test for detecting sensitized lymphocytes from human peripheral blood is described: the poly-L-lysine (PLL) 3400 agglutination test of lymphocytes (PAL test). The incubation of lymphocytes from 30 cancer patients, 30 patients with non-malignant disease and 40 healthy controls with PLL (mol. wt 3400) is evaluated. The test was positive in 83 percent of malignant and 20 percent of non-malignant cases. All healthy controls were negative. Other peptides tested showed no significant difference in reaction between malignant and non-malignant diseases. The mechanism of detection of sensitized lymphocytes from patients with malignant disease through agglutination induced by PLL 3400 is discussed.

Agglutination Tests↗