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Biomedical subjects

G F Ibrahim

Publications and source records attributed to G F Ibrahim.

12 recordsLinked to original sources
PubMed1996

Multiplex PCR assay for the routine detection of Listeria in food.

The development and validation of a multiplex PCR assay for the detection of Listeria that can be employed in routine investigation of food samples are described. The assay, which employs a short culture enrichment step followed by isolation of bacterial cells and detection by multiplex PCR reaction, is highly sensitive and specific for the detection of Listeria monocytogenes and all other Listeria species. Over 350 food samples were tested in parallel by standard cultural procedures and the PCR assay, with no false-positive or false-negative results obtained with the PCR assay. Compared to the standard cultural methods the PCR assay is highly sensitive, cost effective and extremely rapid with results obtained within 48 h from sample receipt.

Food Microbiology↗
PubMed1987

Production of potent polyclonal antibodies to bacterial membrane amphiphiles.

Lipid A (LA), ketodeoxyoctonate (KDO) and lipoteichoic acids (LTA) were used to produce homologous polyclonal antibodies. These haptens were administered to rabbits in differing immunogenic forms, using multiple intradermal and intraperitoneal injections with complete Freund adjuvant. Booster injections were either made intradermally with incomplete Freund adjuvant or intravenously in saline. The immune-response was monitored regularly with an enzyme-immunoassay. Lipid A and KDO covalently linked to bovine serum albumin (BSA), with hapten densities per BSA molecule of 17 and 9, respectively, produced nondetectable immune-response. Acid-hydrolysed and intact cells of Salmonella minnesota Re 595 used as LA and KDO immunogens, respectively, produced significant immune-response when administered intradermally or intraperitoneally. Good immune-response was obtained with LTA covalently linked to BAS. However, a better result was obtained with crude LTA, containing 21.5% proteins. Generally, the lengthy immunization schedules used produced IgG antibodies to the antigens and the highest reciprocal titres attained were 75,000, 55,000 and 150,000 for LA, KDO and LTA, respectively. Meaningful expression of antisera titres by enzyme-immunoassay is discussed. We defined titre as the reciprocal antiserum dilution of the intercept of the mid-point on the linear section ending at 0.2 absorbance on the antiserum dilution curve.

Animals↗
PubMed1985

Immunological relationships between Salmonella flagella and their potential application for salmonellae detection by immunoassay.

Native flagella of ten Salmonella serotypes were shown to possess serotype-specific antigenic determinants as well as a multiple of common antigenic determinants, which varied in concentration. Each common antigenic determinant was shared by certain, but not all, serotypes. This has been demonstrated from agglutination and radioimmunometric assay (RIMA) results, using ten antisera raised in rabbits against purified polymeric flagellins from ten Salmonella serotypes as immunogens. The minimum detectable populations of salmonellae, as determined by RIMA varied considerably, due to variation in concentrations (and possibly types) of common antigenic determinants. However, the results demonstrated the feasibility of utilizing RIMA for the qualitative detection of salmonellae, using a mixture of only the ten antisera and 125I-labelled protein A as a general tracer. In this way, 77 different salmonellae were detected in less than 8 h after culturing in selective broth. The RIMA developed was specific for salmonellae and showed no cross-reactions with high populations of other members within the family Enterobacteriaceae.

Agglutination↗
PubMed1985

Immunological relationships between Salmonella flagellins and between these and flagellins from other species of Enterobacteriaceae.

Ten antisera raised in rabbits, against polymeric flagellins from ten different Salmonella serotypes were used to determine the relative cross-activities between salmonella flagellins in monomeric form. The results showed a high degree of cross-reactivity between the antisera (IgG antibodies) and all monomeric flagellins investigated. Consequently, it was possible to detect the ten Salmonella serotypes, after heat depolymerization of flagella using radioimmunometric assay with only one antiserum raised against polymeric flagellin from one serotype. The results also showed that native flagella differed antigenically from repolymerized and monomeric flagellins. This is possibly due to changes in the tertiary structure of sub-units of flagella when depolymerized with acid or heat resulting in unfolding and unmasking of common antigenic determinants. The unfolded and unmasked antigenic determinants were not only common to the Salmonella serotypes investigated, but also to other members of Enterobacteriaceae.

Antibody Specificity↗
PubMed1985

Rapid detection of salmonellae by immunoassays with titanous hydroxide as the solid phase.

Radioimmunometric and enzyme-immunometric assays were developed for the detection of salmonellae in pure and mixed cultures as well as in 59 food samples. The performances of titanous hydroxide suspension and microtiter plates as the solid phase for the immobilization of microorganisms were compared in these immunoassays. Detection of populations of salmonella cells in pure culture, diluted with saline, was 4- to 10-fold more sensitive with the microtiter plates. However, with mixed culture of salmonella and other enterobacterial species, the detection sensitivity with titanous hydroxide was 100- to 160-fold more sensitive than with microtiter plates. Good correlation existed between results of a standard cultural method for the detection of salmonellae in foods and those obtained from radioimmunometric and enzyme-immunometric assays utilizing titanous hydroxide. However, a high incidence of false-positive and false-negative results with food samples occurred with the enzyme-immunometric assay utilizing microtiter plates. The results provided strong evidence for the merits of substituting titanous hydroxide for microtiter plates as the solid phase for the immobilization of salmonellae for their detection by immunoassays. The immunoassays were rapid and enabled the analysis of a large number of selective enrichment cultures of food samples for salmonellae within 8 h.

False Negative Reactions↗
PubMed1985

Production of potent Salmonella H antisera by immunization with polymeric flagellins.

Highly purified polymeric flagellin preparations from 10 different Salmonella serotypes were used to produce specific Salmonella H antisera with high titers by the immunization of rabbits. Antigen emulsions in complete Freund adjuvant were administered at the rate of 50 micrograms per rabbit by multiple intradermal injection. Booster injections were given 110 days after the primary immunization. The immune response was monitored regularly over a period of 200 days. The results showed that the H titers, determined with 125I-labeled antigens, averaged 61,000 +/- 39,000, and the H agglutination titers of 83% of the animals were greater than 40,000. The high titers of the immunized animals persisted for approximately 4 months. The O agglutination titers of the antisera were less than 10 for 9 of the flagellin preparations and ranged from 10 to 320 for the remaining preparation. The antisera obtained were serotype specific after appropriate dilution.

Agglutination Tests↗
PubMed1985

Immobilization of microorganisms for detection by solid-phase immunoassays.

Several cultures of gram-negative and gram-positive bacteria were successfully immobilized with titanous hydroxide. The immobilization efficiency for the microorganisms investigated in saline and broth media ranged from 80.2 to 99.9%. The immobilization of salmonellae was effective over a wide pH range. The presence of buffers, particularly phosphate buffer, drastically reduced the immobilization rate. However, buffers may be added to immunoassay systems after immobilization of microorganisms. The immobilization process involved only one step, i.e., shaking 100 microliter of culture with 50 microliter of titanous hydroxide suspension in polystyrene tubes for only 10 min. The immobilized cells were so tenaciously bound that vigorous agitation for 24 h did not result in cell dissociation. The nonspecific binding of 125I-labeled antibody from rabbits and 125I-labeled protein A by titanous hydroxide was inhibited in the presence of 2% gelatin and amounted to only 5.6 and 3.9%, respectively. We conclude that this immobilization procedure is a potentially powerful tool which could be utilized in solid-phase immunoassays concerned with the diagnosis of microorganisms.

Bacteriological Techniques↗
PubMed1985

Method for the isolation of highly purified Salmonella flagellins.

Ten different Salmonella serotypes were grown in a chemically defined medium supplemented with 0.01% yeast extract. After sedimentation of the cells by centrifugation, flagella were detached by exposure to pH 2 for 30 min at room temperature. The flagellaless cells were removed by centrifugation, and the flagellin in the supernatant was further purified by high-speed centrifugation, ammonium sulfate precipitation, and dialysis in 50,000-molecular-weight-cutoff tubing. The 10 flagellin preparations were of a high degree of purity, as demonstrated by electron microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and measurement of salmonella H and O agglutination titers of antisera raised in rabbits with the flagellin preparations as immunogens.

Antigens, Bacterial↗
PubMed1985

Automation and limitation of 17 beta oestradiol dehydrogenase in monitoring ovarian function.

An automated enzymatic assay which measures combined concentrations of urinary oestrone and oestradiol within 15 minutes of hydrolysis has been developed using placental 17 beta oestradiol dehydrogenase. This assay is comparable to radioimmunoassay in its precision and sensitivity and is quicker, more convenient, and more economical. The 17 beta oestradiol dehydrogenase assay was used to monitor ovarian response in infertile women being treated through stimulated ovarian cycles or in vitro fertilisation programmes. Because the assay did not respond to oestriol it was less reliable than total urinary oestrogens in monitoring ovarian responses, particularly during treatment with drugs which enhanced oestriol excretion. In natural ovulatory cycles rising 17 beta oestradiol dehydrogenase values preceded peak luteotropin concentrations by up to 18 hours and improved detection of ovulation. The 17 beta oestradiol dehydrogenase assay processes large numbers of urine samples quickly and economically and enables accurate monitoring of oocyte maturity and ovulation.

17-Hydroxysteroid Dehydrogenases↗
PubMed1983

Rapid acetylcholinesterase screening test for neural tube defect.

A 5-min optimized automated assay for measuring acetylcholinesterase (AChE) and total cholinesterase (ChE) in amniotic fluids is described in which a centrifugal analyzer is used. Total ChE was measured by using acetylthiocholine iodide and monitoring the color developed after 5,5'-dithiobis(2-nitrobenzoic acid) is added. We evaluated four direct and indirect procedures for measuring AChE in which thiocholine is generated. These methods involve either the specific enzyme inhibitors ethopropazine and BW284C51 or the specific substrates S-butyrylthiocholine and acetyl-beta-methylthiocholine. We found the last method distinguishes normal pregnancies most efficiently from those with neural tube defect. Filtering samples through 0.22-micron membrane filters before analysis decreased the incidence of false positives. Significantly increased AChE concentrations were noted in all abnormal pregnancies (n = 45) and in 4.8% of normal pregnancies (n = 310). The rapid AChE assay involving acetyl-beta-methylthiocholine, when applied alongside a rapid alpha-fetoprotein assay, increases the efficiency of screening programs for pregnancies with neural tube defect to 99.4%. It also identifies samples with negative results for alpha-fetoprotein that require further examination by the sensitive gel-electrophoretic test for AChE.

Acetylcholinesterase↗
PubMed1980

Determination of staphylococcal enterotoxin A in cheddar cheese produced without starter activity.

Three variants of the chloramine-T radioiodination method were used to iodinate staphylococcal enterotoxin A with 125I. Only one method consistently produced usable labels for radioimmunoassay. The iodine incorporation was 55 to 76%; the specific activity was 3.5 to 5.5 muCi/microgram of enterotoxin, and the label was extremely stable on storage at -20 degrees C. Determinations of the enterotoxin in extracts of cheddar cheese produced without starter activity were carried out with the radioimmunoassay system and protein A as antibody immunoadsorbent. The assay buffer used in this system significantly influenced the detected levels of enterotoxin in the cheese extracts. Phosphate buffer, but not tris(hydroxymethyl)aminomethane (Tris) buffer, caused gelling of cheese extract proteins, thus resulting in an incomplete separation of free from antibody-bound 125I enterotoxin. When Tris buffer was used, the results indicated a high degree of accuracy and precision for this radioimmunoassay. The lowest detectable enterotoxin concentration in cheese extract was 0.5 ng/ml.

Animals↗