Conventional Agglutination Test Research Articles

Development of colloidal gold immunochromatographic strips for detection of Riemerella anatipestifer.

Riemerella anatipestifer is one of the most important bacterial pathogen of ducks and causes a contagious septicemia. R. anatipestifer infection causes serositis syndromes similar to other bacterial infections in ducks, including infection by Escherichia coli, Salmonella enterica and Pasteurella multocida. Clinically differentiating R. anatipestifer infections from other bacterial pathogen infections is usually difficult. In this study, MAb 1G2F10, a monoclonal antibody against R. anatipestifer GroEL, was used to develop a colloidal gold immunochromatographic strip. Colloidal gold particles were prepared by chemical synthesis to an average diameter of 20±5.26 nm by transmission electron microscope imaging. MAb 1G2F10 was conjugated to colloidal gold particles and the formation of antibody-colloidal gold conjugates was monitored by UV/Vis spectroscopy. Immunochromatographic strips were assembled in regular sequence through different accessories sticked on PVC plate. Strips specifically detected R. anatipestifer within 10 min, but did not detect E. coli, S. enterica and P. multocida. The detection limit for R. anatipestifer was 1×106 colony forming units, which was 500 times higher than a conventional agglutination test. Accuracy was 100% match to multiplex PCR. Assay stability and reproducibility were excellent after storage at 4°C for 6 months. The immunochromatographic strips prepared in this study offer a specific, sensitive, and rapid detection method for R. anatipestifer, which is of great importance for the prevention and control of R. anatipestifer infections.

A New Superagglutination Test to Minimize False Negative and False Positive Results Common with Plate/Slide Agglutination Tests for the Diagnosis of Infectious Diseases

Agglutination is the clumping of antigenic particles by antibodies. Several diagnostic kits for infectious diseases of animals and humans, based on slide agglutination/plate agglutination tests like the Rose Bengal Plate Test ( RBPT) for diagnosis of brucellosis are used worldwide. False negative and false positive results are commonly encountered in these conventional agglutination tests. Simple, cost effective modifications can help in circumventing these problems. The novelSuperagglutinationTest reported here is a modified slide / plate agglutination test. False negative results due to smaller clumps formed by low titer of antibodies in serum are minimized by the addition of biotinylated antiglobulin followed by Avidin which forms easily detectable larger clumps. Similarly, prior staining of serum antibodies with a dye helps in differentiating a specific agglutinate formed by both antigen and antibody, from a non -specific aggregate of antigen alone that leads to false positive results.Superagglutinationis more sensitive than the current agglutination tests and agglutination based diagnostic kits. The modification steps are easy to perform and give a more accurate diagnosis without much increase in the cost of

Fluorescence Polarization Assay for the Diagnosis of Anti-Brucella abortus Antibodies in Cattle Serum: Adaptation for its Use in Microplates and Comparison with Conventional Agglutination Tests

Fluorescence Polarization Assay for the Diagnosis of Anti-Brucella abortus Antibodies in Cattle Serum: Adaptation for its Use in Microplates and Comparison with Conventional Agglutination Tests Bovine brucellosis, which is endemic in Argentina, is controlled by vaccination and slaughter of infected cattle. Conventional agglutination tests and primary binding assays like ELISA and fluorescence polarization assay (FPA) are used for the identification of infected cattle. The FPA has many advantages over the agglutination tests, however most accredited laboratories still use the conventional agglutination tests. FPA has been extensively evaluated in its original format of 10mm x 75mm glass tubes, while there are little reports on its performance in the 96-well microplate format. The aim of the present study was to set the conditions for the use of a commercially available antigen (the O-polysaccharide from B. abortus 1119-3 conjugated with fluorescein isothiocyanate) for the FPA assay in a 96-well microplate format, and to compare its diagnostic performance with the conventional agglutination tests currently used in Argentina. Serum samples were obtained from 149 cows and 20 bulls belonging to free and infected herds from different regions of Argentina. Two dilutions of serum and antigen were assayed and the fluorescence polarization was detected with a Beckman DTX 880 multimode reader.

Serological profile of buffalo (Bubalus bubalis) female calves vaccinated with standard Brucella abortus strain 19 vaccine using rose bengal, 2-mercaptoethanol and complement fixation tests

The serological profiles of 21 female buffaloes vaccinated between 3 and 8 months of age using Brucella abortus strain 19 (S19) were evaluated by rose bengal (RBT), 2-mercaptoethanol (2ME) and complement fixation (CFT) tests. The serum strains were collected in day zero, 15, 30, 45, 60th days and subsequently to each 30 months, until 720th day after vaccination. No animal showed reaction in day zero. In 15th day above 95% of animals revealed reaction in all tests. All the animals presented absence of reactions in CFT, RBT and 2ME tests at 270, 300 and 360 days after vaccination, respectively. Our finding highlighted early response in CFT compared than other conventional agglutination tests. None of animals presented oscillation of titers or reactions in any test after 360 day of study, which enables the use of these tests after this period without interference of antibodies from S19 vaccine origin between 3 and 8 months in buffalo heifers.

輸血歴がない初回妊娠（双胎）後期に産生された抗Ｄ＋Ｃ抗体による新生児溶血性疾患の一例

We report a case of Rh-incompatible hemolytic disease of bizygotic twin new borns delivered from a Rh D and C-negative woman without a history of prior pregnancy or blood transfusion.The alloimmune hemolysis was caused by maternal anti-D (first baby) and anti D+C (second baby) alloantibodies, which were detected at 32 week's gestation by gel tests (Micro Typing System) and at 34 weeks by conventional agglutination tests with glass tubes. The former method was more sensitive than the latter for detecting red cell alloantibodies.This case suggests that hemolytic disease of the newborn can occur during a first pregnancy even in a woman without prior blood transfusion.

A New Solid-Phase Method for ABO Grouping, Rh Phenotyping, and Kell Determination

Background: In comparison with a well established agglutination method, a newly developed solid-phase method for ABO grouping, Rh phenotyping, and Kell determination was examined for its suitability in routine testing. This new method is characterized by the use of monoclonal IgM antibodies of blood group specificities which are immobilized onto the polystyrene microplate wells during the test procedure. Red blood cell suspensions are added to the prepared wells and are allowed to react immunologically with the solid phase. Positive reactions are indicated by the adherence of red blood cells over the entire surface of the wells, whereas negative reactions form discrete red blood cell buttons in the center of the wells. The uniformity of the reaction patterns permits an objective reading of the results, both visually and spectrophotometrically. Material and Methods:Blood samples of 631 healthy blood donors and 237 patient samples were determined by the solid-phase method, and the results were compared with those obtained by the conventional agglutination test. In addition, a panel of weak A and B subgroups and D variants was determined. Results:Determining the ABO blood groups as well as the Rh phenotypes and the Kell antigen, we found complete accordance between both methods. Detecting A and B subgroups and D variants, the solid-phase method was found to be more sensitive when compared with the agglutination method. Conclusions:Due to the ease of handling of the new solid-phase assay and the unequivocal test results, the method is suitable for manual routine testing in small- to medium-sized laboratories as well as for automation. The reaction patterns can easily be interpreted visually or by computer software-supported readers. Storing the microplates in a refrigerator, the results remain stable for several days.

The levels of memory (CD45RA-, RO+) CD4+ and CD8+ peripheral blood T-lymphocytes correlate with IgM rheumatoid factors in rheumatoid arthritis.

We investigated whether, in rheumatoid arthritis (RA), the CD45 isoform expression of peripheral blood T-lymphocytes (T-PBL) is related to auto-immune processes (e.g. IgM rheumatoid factors) and to clinical manifestations. By three-colour flow cytometry, we quantified three subsets of CD4+ or CD8+ T-PBL: "naive" CD45RA+,RO-, "transient" CD45RA+,RO+, and "memory" CD45RA-,RO+ cells, in 102 patients with RA and in 41 age- and sex-matched controls. The serum levels of rheumatoid factors (RF) were determined--besides conventional agglutination tests--by ELISA (IgM-RF). Extensive clinical examination was performed at the time of blood sampling. In RA, age, sex and drug therapy did not constitute major influences on the CD45RA/RO patterns. In "healthy" men, higher age significantly' correlated with fewer naive and more memory CD4+ T-PBL (P < 0.01). In RA, distinct correlations between the T-PBL subsets, autoimmune and clinical manifestations became obvious when patients with low and high levels of RF against human IgG Fc fragments, as determined by ELISA, were analysed separately. RA patients with high IgM-RF had elevated proportions of CD45RO+ T-PBL (P < 0.05), that correlated with clinical parameters of disease activity (tender joint count, Ritchie index, P < 0.05) and outcome (Health Assessment Questionnaire, Larsen radiographic scores, P < 0.05). The proportions of memory CD4+ and CD8+ T-PBL correlated strongly (P < 0.001) with the IgM-RF levels. Within 1 year, only three of 34 patients (disease duration of 5-9 years) showed seroconversion from low to high levels of IgM-RF (and positive agglutination tests); this was paralleled by reductions in naive and increases in transient T-PBL (P < 0.02). Thus, in RA, the proportions of memory CD4+ and CD8+ T-PBL correlate with the level of IgM-RF and, together with transient T-PBL, with clinical parameters of disease activity and outcome.

Highly sensitive detection of fungal antigens by ultrasound-enhanced latex agglutination.

Treatment with ultrasound has been employed to greatly enhance the sensitivity of commercially available latex agglutination tests for fungal antigens. This 5 min procedure detects 40 pg ml-1 of Candida albicans mannan and 70 pg ml-1 of Aspergillus fumigatus galactomannan, a 250 and 500-fold improvement respectively over conventional agglutination test sensitivities. The ultrasound-enhanced test offers the possibility of improved diagnosis and management of patients with systemical candidosis or invasive aspergillosis.

RAPID GM1 GANGLIOSIDE LATEX AGGLUTINATION SLIDE TEST FOR CHOLERA TOXIN

In conventional latex-particle agglutination tests, latex beads are either coated with antibody and clump if antigen is present in a sample or are coated with antigen and clump if an antibody is present in a sample. Ligands other than antigen and antibody have not been used previously for latex-particle agglutination tests. We report here a new variation, a sandwich latex-particle agglutination test in which interactions between GM1 gangliosides, antigen (cholera toxin) and anticholera antibody are used to affect agglutination. the principles that are described can be applied to develop tests for the rapid detection of other small ligands.

DIPSTICKS FOR DETERMINING ABO BLOOD GROUPS

Dipsticks for determining ABO blood groups were developed, based on the principles of dot immunobinding assays. Their sensitivity and specificity equalled those of conventional agglutination tests and they were simple, fast, stable, inexpensive, and easy to interpret. Since they used whole blood and did not require refrigeration or equipment, they should be useful for determining blood groups away from the hospital setting.

Rapid Identification of Colonization Factor Antigens (CFA/I and CFA/II) of Enterotoxigenic E. coli (ETEC) and O-Antigens of Enteropathogenic E. coli (EPEC) by Coagglutination Test

Specific antisera for CFA/I (O78), CFA/II (O6) or enteropathogenic (EPEC) somatic Oantigens (serogroups O26, O86a and O111) were adsorbed to Staphylococcus aureus (strain Cowan I) to produce coagglutination reagents. Conventional agglutination tests with bacterial cells showed that antiserum against strain C922a-1 reacted with strain C91f, whereas anti-C91f did not agglutinate cells of strain C922a-1. Antisera raised against strains 4334f and C91f agglutinated their bacterial cells. Anti-303d did not react with strains C922a-1, 91f, nor with 4334f. Similar results were obtained with tests performed with COA-reagents of the above strains. Homologous antisera diluted 1: 1000 gave positive COA reaction, after being adsorbed to Cowan I. Such diluted antisera gave negative agglutination reaction in the conventional agglutination tests. The COA test is at least 100 times more sensitive than the conventional agglutination test. Hence, preparation of COA reagents implies practically economical use of antisera. Furthermore, the COA technique is much more sensitive and rapid (< 5 min) than immundiffusion test which takes ≥ 24 h to read and in which large molecular weight antigens fail to diffuse in to agarose. 30 CFA/I strains (100%) gave COA positive reaction and only 25 CFA/II strains (71,4%) were positive with CFA/II COA reagents. The coagglutination technique was also applicable to identification of enteropathogenic E. coli by using somatic O-antigens (026, 086a and Olli) antisera. Sensitized Staphylococcus aureus (Cowan I) by EPEC O-antigen sera diluted to ≥ 1/1500 could specifically aggregate strains belonging to corresponding O-serog-roup. In conclusion the COA technique is an appropriate, rapid diagnostic tool which can easily be used for epidemiological studies in developing and industrialized countries.

Stained Bacterial Antigens for Use in Microagglutination Procedures

Abstract We describe the preparation of tetrazolium-stained antigens from three significant bacterial fish pathogens — Aeromonas hydrophila, A. salmonicida, and enteric redmouth bacterium — for use in a microagglutination system. Serum titers revealed by the microagglutination test and the conventional tube agglutination test were practically identical. The principal advantages of the microagglutination procedure relate to conservation of time, space, and seologic reagents.

An immunological study of the pili of Pseudomonas aeruginosa.

An attempt was made to correlate serological relationships determined by the pili, the flagella and the O-somatic antigens of Pseudomonas aeruginosa, and to make a preliminary assessment of use of the pilus antigen as an epidemiological marker. A method is described for the preparation of antiserum specific for the "normal' PSA pili of P. aeruginosa. A high titre of pilus antibodies was obtained by immunizing rabbits with mutants whose pili had lost their ability to retract into the cell. The "normal' form of the organism, with retractile pili, was poorly agglutinated by high-titre anti-pilus serum, but suspensions of it that had been treated with osmium tetroxide showed greatly increased agglutinability. Antibody labelling for electron microscopy was used to determine the serological relations of pili and of flagella for P. aeruginosa strains belonging to different serological groups as defined by O-somatic antigens. The distribution of pilar and flagellar antigens among strains was not correlated with the O-somatic serotype. A strain of P. aeruginosa carrying a drug-resistance plasmid had fewer "normal' PSA pili than the background strain.

SEROLOGICAL INVESTIGATIONS IN SUSPECTED BRUCELLOSIS

Sera from over 200 hospital patients were examined for brucella antibodies by conventional agglutination tests and by newly suggested methods. Since these new methods mainly depend on the detection of microglobulins (IgG) which reflect activity of the infection, they have been found to be of particular help in the clinically doubtful case of the disease. Sera from healthy adults and children or from uninfected hospital patients rarely possess these antibodies except in low titre. Detailed results are given for 8 out of 16 cases in adults and 8 of 12 acute cases in children. Complement-fixation and anti-human-globulin tests may usefully be employed in surveillance of patients after apparent recovery from an acute attack of the infection. We have confirmed that in some cases of chronic brucellosis these tests are indispensable.

Serologically specific lipopolysaccharides from oral Veillonella

Biologically active (pyrogenic, Schwartzmann-reactive) lipopolysaccharides were prepared by phenol-water extraction of nineteen strains of oral anaerobic gram-negative cocci conforming to the characteristics of the genus Veillonella. Analyses of certain of these lipopolysaccharides for lipid, protein, polysaccharide, N and P contents revealed a striking similarity in gross chemical composition. Conventional agglutination tests and haemagglutination tests employing lipopolysaccharide-sensitized sheep erythrocytes compared favourably as a means of determining the potency of anti-veillonella rabbit sera. The lipopolysaccharides appeared to be serologically specific as determined by haemagglutination-inhibition tests, which established several serotypes of veillonellae. This specificity could not be demonstrated by conventional agglutination tests or by agar-gel diffusion tests using cell extracts.

Standardization and stabilization of an extract from Leptospira biflexa and its use in the hemolytic test for leptospirosis.

A previous report from this laboratory (Cox, 1955) described a hemolytic reaction (HL) which involved the ability of leptospiral extracts to sensitize sheep erythrocytes to hemolysis in the presence of leptospiral antiserum and complement. These extracts were similar in preparation to those previously described by Chang and McComb (1954), who described their use in a hemagglutination test. The HL procedure was shown to be broadly specific, in sharp contrast to the high degree of type specificity seen in the conventional agglutination test. In comparison with the HL procedure, the hemagglutination test of Chang and McComb (1954) was found to be broadly specific but considerably less sensitive, a finding subsequently confirmed by Chang, et al (1957). Although the HL reaction as first reported satisfied the demand for a serodiagnostic test which was broadly specific and safe in operation, the procedure for preparation of the HL extract was hazardous in that it was prepared from cultures of Leptospira pomona. Furthermore, the HL reaction as described in the first report was not simplified with respect to standardization of reagents for performance as a routine procedure. Subsequent studies in this labora-

A serological study of Pasteurella haemolytica.

It has been shown that strains of Pasteurella haemolytica possess a soluble capsular substance which is a polysaccharide. A haemagglutination test is described in which the soluble polysaccharide was adsorbed to type O human red cells. These treated red cells were agglutinated in the presence of appropriate dilutions of specific immune sera. Fifty-one strains of P. haemolytica isolated from cases of shipping fever in Canada were found to be serologically homogeneous by means of the haemagglutination test. Strains of P. haemolytica from the U.S.A., Great Britain, and Europe were found to be serologically identical with the Canadian strains by the haemagglutination and conventional agglutination test.

Estimation of Vi antibody employing erythrocytes treated with purified Vi antigen.

Summary1. The preparation of a sensitive and specific Vi agglutination reagent by sensitization of human type O erythrocytes with Vi antigen isolated from E. colt has been described. 2. Vi antibody content of rabbit and human sera was determined by this method and the conventional bacterial agglutination test. The hemagglutination test provided a more sensitive measure of Vi antibody by a factor of 3 to 6 fold. Greater variability in Vi titers was encountered in testing the sera of typhoid carriers; however, both methods were in good agreement in the detection of Vi antibody in these sera. The advantages inherent in the Vi hemagglutination procedure are discussed.

Escherichia coli hemagglutinin response of adult volunteers to ingested E. coli 055 B5.

SummaryThe E. coli hemagglutinin response of adult volunteers to ingestion of E. coli 055 and a “normal” strain of E. coli, respectively, was determined. 1. Increase in E. coli 055 hemagglutinin titers was observed in all subjects following administration of living organisms in large numbers and in two-thirds of individuals who received smaller numbers. 2. None of the controls who received milk without E. coli showed such an antibody response, nor did the volunteers who ingested large numbers of killed 055 organisms or viable bacteria of a “normal” strain of E. coli. 3. Good correlation was found between increases of E. coli 055 hemagglutinin titers and the appearance of bacterial O agglutinins. 4. The E. coli hemagglutination test proved to be more sensitive than the conventional bacterial agglutination test, inasmuch as the former method with the postfeeding serum specimens yielded antibody titers which were from 5 to 20 times higher and detected antibodies in prefeeding serum specimens which were not d...