Mycoplasma-latex agglutination reaction

Morton, Harry E. (University of Pennsylvania, Philadelphia). Mycoplasma-latex agglutination reaction. J. Bacteriol. 92:1196-1205. 1966.-The building up of Mycoplasma cell mass through adsorption to carrier particles as a method for enhancing the agglutination reaction to identify Mycoplasma is described. Mycoplasma cells of human, avian, swine, goat, sewage, and tissue-culture origin were adsorbed to latex particles (0.81 mu) and then were agglutinated by immune sera. The adsorption was demonstrated by electron microscopy. Either the cells or their antibodies, depending on which came into contact with the latex particles first, were adsorbed. The test, completed in less than 2 hr, consisted of serially diluting immune sera with buffered saline, adding the antigen, incubating in a water bath, centrifuging, and reading the reaction under 50 x microscope magnification. The antigen in each reaction tube, representing the growth from about 1.6 ml of culture, was estimated to contain 23 mug of protein (approximately one-tenth the amount of Mycoplasma cells needed for a direct agglutination reaction). In the sera from rabbits undergoing immunization with Mycoplasma antigens, the presence of anti-Mycoplasma antibodies was detected much sooner in the Mycoplasma-latex agglutination reaction test than in the agar-gel diffusion reaction and the growth inhibition tests. Four different lots of latex particles showed excellent uniformity of behavior and stability during storage and testing.     

Commercially available histoplasmin sensitized latex particles in an agglutination test for histoplasmosis

Summary A commercial preparation of histoplasmin sensitized latex particles was tested in an agglutination test with sera from 50 culturally confirmed cases of histoplasmosis in varying stages of the infection. The reactions were superior to those obtained with collodion agglutination and complement fixation tests in which the antigen histoplasmin was also used. The latex agglutination test with the commercially available antigen is easy to do, and can be done in any laboratory equipped to carry out agglutination tests with the common bacterial antigens. It warrants more extensive trial in the general hospital laboratory as a screening test for histoplasmosis, especially the primary, pulmonary type.     

The use of latex agglutination tests for determining Campylobacter species

A comparison was made between three commercially available latex agglutination tests for the detection of Campylobacter. All tests showed clear agglutination with pure cultures of several Campylobacter strains in both the spiral and coccoid form. The Microscreen test was able to detect 10 times less cells than the Campyslide and Meritec tests. The latex tests were also applied to enrichment broth cultures of chicken products. Sixty-nine per cent of the Campylobacter positive enrichment broth cultures were positive with the Microscreen test. The Meritec test detected 63% of the positive samples. The Campyslide test detected only 15% of the positive samples and often showed non-specific agglutination.     

Specific latex preparations in the etiological diagnosis of suppurative bacterial meningitis

The results of the evaluation of the diagnostic latex preparations Bactigen, manufactured by Wampole Laboratories (USA) and intended for the detection of meningococcal antigens, serogropus A, B, C, Y, pneumococcal polyantigens and type b Haemophilus influenzae antigens in the spinal fluid and blood of patients with meningococcal infection and purulent bacterial meningitides, are presented. The pathological material was studied by traditional methods and by the latex agglutination (LAG) test. 522 LAG tests were made, including 414 tests for meningococcal infection, 60 tests for pneumococcal infection and 48 tests for type b H. influenzae. The results of this study revealed that the latex preparations were highly specific with respect to type b H. influenzae antigens and meningococcal antigens (false positive reactions constituted 0.96%). The simplicity of the test and the rapid techniques making it possible to obtain results within 30-40 minutes indicate good prospects of using the LAG test in laboratory practice.     

A nanoparticle amplification based quartz crystal microbalance DNA sensor for detection of Escherichia coli O157:H7

A quartz crystal microbalance (QCM) DNA sensor, based on the nanoparticle amplification method, was developed for detection of Escherichia coli O157:H7. A thiolated single-stranded DNA (ssDNA) probe specific to E. coli O157:H7 eaeA gene was immobilized onto the QCM sensor surface through self-assembly. The hybridization was induced by exposing the ssDNA probe to the complementary target DNA, and resulted in the mass change and therefore frequency change of the QCM. Streptavidin conjugated Fe(3)O(4) nanoparticles (average diameter=145 nm) were used as "mass enhancers" to amplify the frequency change. Synthesized biotinylated oligonucleotides as well as E. coli O157:H7 eaeA gene fragments (151 bases) amplified using asymmetric PCR with biotin labeled primers were tested. As low as 10(-12)M synthesized oligonucleotides and 2.67 x 10(2) colony forming unit (CFU)/ml E. coli O157:H7 cells can be detected by the sensor. Linear correlation between frequency change and logarithmic number of bacterial cell concentration was found for E. coli O157:H7 from 2.67 x 10(2) to 2.67 x 10(6)CFU/ml.     

Sensitivity of an immunomagnetic-separation-based test for detecting Escherichia coli O26 in bovine feces

The sensitivity of a test for cattle shedding Escherichia coli serogroup O26 was estimated using several fecal pats artificially inoculated at a range of concentrations with different E. coli O26 strains. The test involves the enrichment of fecal microflora in buffered peptone water, the selective concentration of E. coli O26 using antibody-coated immunomagnetic-separation beads, the identification of E. coli colonies on Chromocult tryptone bile X-glucuronide agar, and confirmation of the serogroup with E. coli serogroup O26-specific antisera using slide agglutination. The effective dose of E. coli O26 for an 80% test sensitivity (ED(80)) was 1.0 x 10(4) CFU g(-1) feces (95% confidence interval, 4.7 x 10(3) to 2.4 x 10(4)). Differences in test sensitivity between different E. coli O26 strains and fecal pats were also observed. Individual estimates of ED(80) for each strain and fecal pat combination ranged from 4.2 x 10(2) to 4.8 x 10(5) CFU g(-1). These results suggest that the test is useful for identifying individuals shedding a large number of E. coli O26 organisms or, if an appropriate number of individuals in a herd are sampled, for identifying affected herds. The study also provides a benchmark estimate of sensitivity that can be used to compare alternative tests for E. coli O26 and a methodological approach that can be applied to tests for other pathogenic members of the Enterobacteriaceae and other sample types.     

Occurrence of p-fimbriae in enteropathogenic E coli (EPEC) and in E. coli strains isolated from patients with urinary tract infections]

The aim of this study was to gain knowledge of prevalence of P+ clones among EPEC strains isolated from children with diarrhoea and E. coli strains isolated from urine. Three hundred eighty four E. coli strains isolated from children with diarrhoea were tested. They belonged to 11 serotypes (018, 025, 026, 044, 055, 0111, 0114, 0119, 0124, 0125, and 0128). Nine hundred thirty colonies of E. coli from Mac Conkey's agar plated quantitatively with urine samples of 178 individuals suffering from urinary tract infections were also tested. All strains were assayed by mannose-resistant active haemagglutination test (MRHA) and by slide agglutination using self prepared latex reagent for detection of P fimbriae. Out of 384 E. coli strains tested 122 (31.8%) showed presence of adhesins detected by mannose-resistant active haemagglutination test (MRHA) and in 90 (23.3%) out of all tested strains the presence of P fimbriae was found. The highest percentage of P fimbriae prevalence was found in E. coli belonging to the following serotypes: 018 (in 68.9% strains), 025 (in 29.2% strains), and 0125 (in 25.0% strains). This type of fimbriae was also detected in serotypes 026 (9.1%), 044 (8.7%), 055 (5.6%), and 0119 (in 2 strains out of 5 isolated). Out of 933 colonies of E. coli, isolated from 178 urine samples, 434 (46.5%) colonies gave positive results in MRHA test, including 133 positive in latex test for P fimbriae. These studies showed that for MRHA adhesins, including P fimbriae, a parallel examination of higher number of E. coli was necessary.(ABSTRACT TRUNCATED AT 250 WORDS)     

Release of Vi antigens from Salmonella typhi: implications for virulence and diagnosis

The release of Vi antigens from three clinical isolates of Salmonella typhi was measured by a Vi-specific monoclonal antibody. Large quantities of Vi antigens were detected in the culture supernates from all three strains using either passive latex agglutination or rocket immunoelectrophoresis. Vi antigens were also detected in broth cultures of S. typhi containing about 10(5) cells/ml using the sandwich enzyme linked immunosorbent assay. The significance of this finding in relationship to the virulence and the diagnosis of S. typhi was discussed.     

Single cell level detection of Escherichia coli in microfluidic device

Detection of Escherichia coli K-12 in phosphate buffered saline (PBS) was demonstrated in a Y-channel polydimethylsiloxane (PDMS) microfluidic device through optical fiber monitoring of latex immunoagglutination. The latex immunoagglutination assay was performed for serially diluted E. coli solutions using 0.92-microm highly carboxylated polystyrene particles conjugated with polyclonal anti-E. coli. Pre-treatments such as cell lysis or culturing to enhance the signal were not used. Proximity optical fibers around the view cell of the device were used to quantify the increase in 45 degrees forward light scattering of the immunoagglutinated particles. In order to reduce false positive signals caused by antibodies binding to non-viable E. coli cells or free antigens in solution, target solutions were washed three times, and then the results were compared to non-washing treatments. The detection limit was found to be less than 10 cfu ml(-1) (1 cfu per device) without PBS washing (thus detecting non-viable cells and free antigens), or less than 40 cfu ml(-1) (4 cfu per device) with PBS washing (thus detecting viable E. coli cells only).     

Liposome-based microcapillary immunosensor for detection of Escherichia coli O157:H7

Our group has previously reported a sandwich-based strip immunoassay for rapid detection of Escherichia coli O157:H7 [Anal. Chem. 75 (2003) 4330]. In the present study, a microcapillary flow injection liposome immunoanalysis (mFILIA) system was developed for the detection of heat-killed E. coli O157:H7. A fused-silica microcapillary with anti-E. coli O157:H7 antibodies chemically immobilized on the internal surface via protein A served as an immunoreactor/immunoseparator for the mFILIA system. Liposomes tagged with anti-E. coli O157:H7 and encapsulating a fluorescent dye were used as the detectable label. In the presence of E. coli O157:H7, sandwich complexes were formed between the immobilized antibodies in the column, the sample of E. coli O157:H7 and the antibody-tagged sulforhodamine-dye-loaded liposomes. Signals generated by lysing the bound liposomes with 30 mM n-octyl-beta-D-glucopyranoside were measured by a fluorometer. The detected signal was directly proportional to the amount of E. coli O157:H7 in the test sample. The mFILIA system successfully detected as low as 360 cells/mL (equivalent to 53 heat-killed bacteria in the 150 microL of the sample solution injected). MeOH (30%) was used for the regeneration of antibody binding sites in the capillary after each measurement, which allowed the immunoreactor/immunoseparator to be used for at least 50 repeated assays. The calibration curve for heat-killed E. coli O157:H7 has a working range of 6 x 10(3)-6 x 10(7)cells, and the total assay time was less than 45 min. A coefficient of variation for triplicate measurements was < or =8.9%, which indicates an acceptable level of reproducibility for this newly developed method.     

Comparative Evaluation of Five Serological Methods for the Diagnosis of Sporotrichosis

The diagnosis of sporotrichosis can be time consuming. Serological procedures could facilitate the rapid and accurate diagnosis of this disease. A slide latex agglutination (SLA) test for sporotrichosis was developed and compared with the tube agglutination (TA), complement fixation (CF), and immunodiffusion (ID) tests in the serological study of 80 proven human cases of sporotrichosis representing the cutaneous, subcutaneous, and extracutaneous forms of the disease. In addition, the indirect fluorescent antibody (IFA) technique was applied to 61 case sera. In the SLA test, latex particles sensitized with culture filtrate antigens from the yeast form of Sporothrix schenckii (B 959) detected 94% of the cases, as compared to 96% of the cases detected by the TA test, 68% by the CF test, and 56% by the ID test. The IFA test detected 90% of the 61 cases. The SLA and ID tests were specific, showing no reactions with sera from 86 persons with no disease or with diseases other than sporotrichosis. Because of its sensitivity, specificity, ease of performance, and ability to provide results in 5 min, the SLA test is highly recommended for routine use in the clinical laboratory.     

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.     

Constitutive expression of a native human interferon-alpha 1 gene in Escherichia coli

1. A plasmid for constitutive expression of the human interferon-alpha 1 (hIFN-alpha 1) gene in Escherichia coli is constructed on the basis of the cloning plasmid pBR322 using a strong synthetic promoter, synthetic ribosome binding site and a native hIFN-alpha 1 gene excised from a chromosomal clone. 2. The yield of recombinant hIFN-alpha 1 from E. coli LE392 cells transformed with the expression plasmid pJP1R9-hIFN-alpha 1 is evaluated to be 2-6 x 10(7) U/l bacterial culture for metabolic shaker and 6-8 x 10(7) U/l for fermentor.     

Release of Vi antigens from Salmonella typhi: implications for virulence and diagnosis

Abstract The release of Vi antigens from three clinical isolates of Salmonella typhi was measured by a Vi-specific monoclonal antibody. Large quantities of Vi antigens were detected in the culture supernates from all three strains using either passive latex agglutination or rocket immunoelectrophoresis. Vi antigens were also detected in broth cultures of S. typhi containing about 10⁵ cells/ml using the sandwich enzyme linked immunosorbent assay. The significance of this finding in relationship to the virulence and the diagnosis of S. typhi was discussed.     

Latex Test for Quantitative Determination of Escherichia coli Antibody

A latex agglutination test was developed for assay of anti-Escherichia coli antisera. The test is simple, specific, sensitive, and reproducible.     

Latex agglutination test for diagnosing pneumococcal pneumonia in children in developing countries.

OBJECTIVE--To prepare and assess the sensitivity and specificity of a latex agglutination test specific for the serotype of antigen in diagnosing pneumococcal pneumonia in Gambian children. DESIGN--Comparison of agglutination test specific for serotype with culture of blood and lung aspirates, countercurrent immunoelectrophoresis, and commercial latex agglutination tests in diagnosing pneumococcal pneumonia. Cross reaction studies and investigation of 102 control children to determine specificity of agglutination test specific for serotype. SETTING--General medical ward of Medical Research Council laboratories, The Gambia. PATIENTS--101 Gambian children aged between 2 months and 10 years admitted with severe pneumonia. INTERVENTIONS--Serum samples were boiled and treated with edetic acid, and urine samples were boiled and concentrated 25 times before testing. END POINT--A latex agglutination test specific for the serotype of pneumococcal antigen that is sensitive and highly specific for detecting pneumococcus in the urine of patients with pneumococcal pneumonia. MEASUREMENTS AND MAIN RESULTS--Concentrated urine samples from 16 of the 21 children (76%) with pneumococcal pneumonia established by results of culture of blood or lung aspirates gave a positive result with the agglutination test specific for serotype, whereas only four of the 102 urine samples obtained from control children without pneumonia gave positive results. The serotypes of antigens detected in the urine of children with pneumococcal pneumonia and the serotypes of pneumococci isolated from cultures of blood or lung aspirates were the same in all cases. CONCLUSIONS--When performed on urine samples the agglutination test specific for serotype has a high specificity and is more sensitive than culture of blood or lung aspirates, commercial agglutination tests, or countercurrent immunoelectrophoresis in identifying pneumococcal pneumonia. It is easy to use and should be especially useful in communities with limited laboratory facilities.     

Altered colonizing ability for mouse large intestine of a surface mutant of a human faecal isolate of Escherichia coli

Escherichia coli F-17 Sr a human faecal isolate, is resistant to the T-series of bacteriophages (i.e. T2 to T7). A T2-sensitive mutant of E. coli F-17 Sr was isolated following acriflavin treatment. This mutant, E. coli F-17 Sr Ts was found to be sensitive to the entire T-series of phages. E. coli F-17 Sr and E. coli F-17 Sr Ts did not differ quantitatively in total LPS content. However, analysis of LPS revealed that a large fraction of E. coli F-17 Sr Ts was devoid of O-side-chains. This accounted for the sensitivity of this strain to bacteriophages T3, T4, and T7. In addition, E. coli F-17 Sr Ts contained only about half the amount of capsular material contained by E. coli F-17 Sr accounting for the sensitivity of the mutant to bacteriophages T2, T5, and T6. Although the two strains colonized equally well when fed individually to streptomycin-treated mice, when fed simultaneously to streptomycin-treated mice, E. coli F-17 Sr Ts colonized at a level of about 1 x 10(8) cells (g faeces)-1, whereas E. coli F-17 Sr colonized at only 1 x 10(4) cells (g faeces)-1. These studies suggest that bacterial cell surface components modulate the large intestine colonizing ability of E. coli F-17 Sr in the mouse large intestine.     

Growth and survival of E. coli O157:H7 during the manufacture and ripening of a smear-ripened cheese produced from raw milk

AIMS: The behaviour of Escherichia coli O157:H7 was studied during the manufacture and ripening of a smear-ripened cheese produced from raw milk. METHODS AND RESULTS: Cheese was manufactured on a laboratory scale using milk (20 l) inoculated with E. coli O157:H7, and enumeration was carried out using CT-SMAC. From an initial level of 1.52 +/- 0.03 log cfu ml-1 in the milk (34 +/- 2 cfu ml-1), the numbers increased to 3.4 +/- 0.05 log cfu g-1 in the cheese at day 1. During ripening, the numbers decreased to <1 cfu g-1 and <10 cfu g-1 in the rind and core, respectively, after 21 days, although viable cells were detected by enrichment after 90 days. The presence of E. coli O157:H7 in the cheese was confirmed by latex agglutination and by multiplex PCR. CONCLUSION: The results indicate that the manufacturing procedure encouraged substantial growth of E. coli O157:H7 to levels that permitted survival during ripening and extended storage. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of low numbers of E. coli O157:H7 in milk, destined for raw milk cheese manufacture, could constitute a threat to the consumer.     

Susceptibility of common voles to experimental toxoplasmosis

Common voles (Microtus arvalis) in groups of nine to 10 animals were inoculated per os with a dose of 1, 10, 1x10(2), 1x10(3), and of the K1 strain of Toxoplasma gondii. All the common voles inoculated with 1 to 1 x 10(3) oocysts remained subclinical and survived. Three of the 10 voles inoculated with 1 x 10(4) oocysts died between days 7 and 12 post inoculation (p.i.). Antibodies were demonstrated in all the infected voles killed on day 60 p.i. The highest antibody titres in voles detected by the dye test (DT) and latex agglutination test (LAT) were 1,024 and 1,280, respectively.