A rapid chemiluminescent enzyme-linked immunosorbent assay for cytomegalovirus immunoglobulin G antibodies using instant photographic film

A rapid and convenient chemiluminescent enzyme-linked immunosorbent assay (ELISA) for IgG antibodies to cytomegalovirus has been developed which uses low cost equipment. Assays were carried out on transparent microtitre plates and used an anti-human IgG horseradish peroxidase conjugate. Bound peroxidase was detected chemiluminescently using a p-iodophenol-luminol-peroxide reagent. Light emission from the wells of the microtitre plate was detected on instant photographic film (ASA 20,000) held in a specially designed shutter type camera. The semi-quantitative technique was tested in a routine laboratory for a period of 7 wk and the results obtained compared well (95.3% agreement) with those obtained by a conventional colorimetric ELISA using an alkaline phosphatase label.     

A modification of Hepatest, using the Terasaki plate, for the detection of HBSAg in blood donors.

A simple modification of Hepatest (Wellcome Reagents) is described, which lends itself to the rapid large-scale screening of blood donors. The technique uses 4 mul of HBSAb coated turkey cells (Cayzer et al., 1974), and Terasaki plates (Hopkins and Das, 1973), in contrast to the 25 mul used in the microtitre tray technique. This results in a considerable saving on the cost of reagents, the cost per test being reduced to 16% of the cost of the recommended method. In our hands, this modification is as convenient to use as the manual microtitre tray technique. Positive sera from the screen test are also tested for non-specificity using horse IgG coated turkey control cells. The falso positive screen test rate has been reduced by the adoption of a modified buffer containing turkey serum.     

A simple rapid technique to measure neutrophil or serum bactericidal activity

A rapid and simple technique to measure both serum and neutrophil bactericidal activity is described. It is based on microtitre equipment and avoids the cumbersome and time consuming serial dilution and plate counting of conventional experiments. A constant concentration of neutrophils and/or serum is incubated with a series of dilutions of bacterial suspensions. Bacterial survival is estimated at various times from their ability to form colonies on agar and the results conveniently expressed as the largest bacterial population that can be eliminated by either neutrophils or serum alone. The technique is as simple and quick as a standard microtitre MBC test commonly used for antibiotics and scoring of the results takes only a few seconds per sample. Data are presented showing the close correlation between results obtained using this technique and those from conventional tests of neutrophil bactericidal activity and phagocytosis-associated chemiluminescence.     

The SIMULTEST approach for testing mutagens in the Salmonella microtitre fluctuation assay

The concept of combining several histidine-dependent Salmonella strains in a single test, the SIMULTEST, has been applied to the microtitre fluctuation test. The activity of five mutagens was determined in strains TA97, TA98, TA100, and TA102 individually as well as in a SIMULTEST mixture. All five compounds were mutagenic in the SIMULTEST, demonstrating the utility of this time and labour-saving approach of combining strains for testing with this method. The microtitre fluctuation SIMULTEST results were quantitatively comparable to those of the SIMULTEST Salmonella/microsome plate test. The microtitre fluctuation test compared with the plate incorporation assay generally showed more favourable "sensitivity" and "quantity" indices in that four of the five chemicals tested in the fluctuation test were mutagenic at lower doses than in the plate test.     

Development and evaluation of a miniaturised method as an aid to the identification of clinically important anaerobic bacteria

A miniaturised method for the identification of anaerobic bacteria is described which employs microtitre fermentation tests, a spot indole test and a nitrate reduction disc test. The results obtained are directly comparable with those produced by a standard conventional method in use at present.     

A solid-phase immunosorbent technique for the rapid detection of rubella IgM by haemagglutination inhibition.

The rubella haemagglutination inhibition (HAI) test has been modified for the detection of rubella-specific IgM. The rubella HAI test is performed in microtitre plate wells in which IgM from patients' sera has been selectively retained by anti-human IgM bound to the polystyrene surface. The test requires only anti-human IgM-coated microtitre plates, in addition to the standard rubella HAI reagents. The results obtained in this test, a solid-phase immunosorbent technique (SPIT), are in good agreement with results obtained by the sucrose density gradient centrifugation technique (DGCT). Advantages include the essentially unrestricted number of sera which can be tested daily, the availability of results within 24 h and the lack of interference by rheumatoid factor and by rubella-specific IgG.     

Metronidazole resistance test in vitro in strains of Trichomonas vaginalis isolated from clinical material. 2. Proposal for a standard tests]

The purpose of this paper is to recommend a dependable susceptibility assays for detection of resistance to metronidazole in Trichomonas vaginalis, suitable for routine use in clinical and public health care laboratories. Two different assays, the microtitre plate test based on Meingassner's technique and an alternative tube-assay, were scrutinized and compared using 10 metronidazole-resistant isolates from Czechoslovakia, other European countries and USA and 10 drug-susceptible strains isolated in Czechoslovakia. The minimal lethal concentrations of metronidazole determined for the resistant strains ranged from 25 to 317.5 micrograms.ml-1 while those of drug-susceptible from 3.1-12.5 micrograms.ml-1 metronidazole. Results obtained by both assays were highly reliable and mutually comparable. The authors recommend the microtitre plate test as a method of choice.     

Application of microtitre plates and fluorescence reading to shorten handling of Phadezym RAST® and Phadezym IgE PRIST®

To enable shorter and more convenient testing, the Phadezym RAST® and Phadezym IgE PRIST® procedures for the determination of specific and total IgE were modified in three ways: (i) allergen-coupled paper discs were tested in microtitre wells; (ii) the incubation times were reduced to 1 hr with serum and 2 hr with the anti-IgE by shaking the plates at room temperature; and (iii) the fluorogenic substrate used reduced the development time to 15 min. Determination of IgE antibody specific for fifteen inhalation allergens by the modified fluorescence test (FEIA) and by the conventional Phadezym RAST® (EIA) was performed on the serum of thirty-two patients suffering from asthma/rhinitis: correlation studies for these sera showed that 96.1% of the results fell in the same class. In these patients, both FEIA and EIA detected the same proportion of skin-prick tests (SPT) positive results (67%). With the FEIA, 4/165 (2.4%) class 1 results were found in eleven non-atopic subjects (symptom free, fifteen negative SPT, total IgE lower than 80 kU/1), compared to 1/165 (0.6%) with the EIA. In twenty cord sera, both FEIA and EIA found 4/300 (1.3%) class 1 results. For the determination of total serum IgE, the microtitre FEIA showed a detection limit of 0.5 kU/1 and an excellent correlation with Phadezym IgE PRIST® (n= 66 serum, r= 0.99). These data indicate that the adaptation of Phadezym RAST® and Phadezym IgE PRIST® to microtitre plates and fluorescence technology has resulted in a time-saving and easy to perform within-day assay which provided results as reproducible, sensitive and specific as those of the conventional procedure.     

Simplified method for the rubella haemagglutination inhibition screening test.

A modification of the rubella haemagglutination inhibition (HAI) test in which both pretreatment and serum titration are carried out in wells of the same microtitre plate saves time, labour, and materials and gives results comparable to a conventional HAI procedure.     

Microtitre plate measurement of platelet response to hypotonic stress.

The conventional method of assessing the platelet response to hypotonic stress (HSR) was adapted to allow microtitre plate technology to be used. After water is added to a platelet suspension two sequential readings are taken at 414 nM on a vertical microplate reader. The difference between the second (three minutes) and the first (one minute) was defined as the HSR. This method allowed the relation between platelet concentrate pH and viability to be confirmed, and an HSR value for use in quality control was established. The method correlated well with the conventional technique and permitted measurement of undiluted samples as well as of products with a high free haemoglobin concentration.     

Evaluation of a modified fluorescent technique for the detection of Australia antigen in liver tissue

A modified fluorescent method is described for the detection of Australia antigen (AuAg, hepatitis associated antigen, hepatitis B antigen) in liver tissue and the results are compared with those obtained with the conventional technique. Of 84 unselected biopsies tested, 40 were positive by the conventional as well as by the modified method. Seven additional biopsies, negative by the conventional method, became positive by the latter technique. Moreover, the intensity of the fluorescence and the number of positive cells were clearly increased in 85% of the specimens which were positive by the conventional technique. The modified method proves to be more sensitive than the conventional immunofluorescent procedure.     

Use of human embryo lung fibroblasts to detect a heat labile toxin of Escherichia coli from children.

In order to detect the heat labile toxin of Escherichia coli human embryo lung fibroblast cells were seeded with whole cell lysate preparations of the organism to be tested. Positive results consisted of growth inhibition and cytopathic change which were easily seen. Heat-labile toxin was produced by strains belonging to the conventional epidemic serotypes of E. coli (EEC) and by non-EEC strains. Toxin-producing organisms were detected in 20% of healthy children examined. The method is suitable for use in the diagnostic laboratory and is easily adapted to a microtitre plate system suitable for screening purposes.     

A comparison of immunocytochemical staining enhancement methods using a rapid microtitre immunocytochemistry assay (MIA)

A method is described which allows rapid and quantitative comparison of immunocytochemical staining procedures. Cells grown and fixed in microtitre plates are probed with increasing dilutions of primary antibody and then stained using the procedures under test; the resulting staining intensities are determined using a microtitre plate reader. The microtitre immunocytochemistry assay (MIA) has been used to compare the sensitivities of enhancement procedures based on immunoperoxidase and immunogold staining. Silver enhancement of DAB staining was found to be the most sensitive technique giving up to 200 fold amplification of the peroxidase staining.     

Modification of the Minitek Miniaturized Differentiation System for characterization of anaerobic bacteria.

The Minitek Miniaturized System (BBL) was modified for characterization of anaerobic bacteria. The modified system and the conventional Center for Disease Control method were used to test a variety of anaerobic bacteria, and results were compared. Tests performed by both techniques were indole and H2S production, esculin hydrolysis, nitrate reduction, and fermentation of glucose, mannitol, lactose, sucrose, maltose, salicin, glycerol, xylose, arabinose, mannose, rhamnose, and trehalose. The manufacturer's recommended procedure for the Minitek system was modified by using a new suspension medium (Lombard-Dowell broth) and an inoculum equivalent to the density of a McFarland no. 5 nephelometer standard. The Minitek results, recorded after 48 h, agreed satisfactorily with the conventional test results, usually recorded after 5 to 7 days of incubation. In the examination of 80 strains representing 22 different species or subspecies of anaerobic bacteria, with 16 biochemical tests performed in triplicate, 93.8% of the Minitek test results agreed with those of the corresponding conventional tests. Only tests for indole, H2S, and nitrate reduction gave less than 90% agreement. It was concluded that the modified Minitek system is a suitable substitute for the more expensive and time-consuming conventional procedure for determining carbohydrate fermentation and esculin hydrolysis by anaerobes. This system, when used in conjunction with other tests, can effectively aid in the definitive identification of commonly isolated anaerobes.     

'RETCIF': a rapid, sensitive method for detection of viruses, applicable for large numbers of clinical samples

Rapid detection of viruses in clinical samples is important for continuing appropriate antiviral treatment and discontinuing unnecessary antibacterial treatment, as well as for excluding viral pathogens. Yet detection of viral agents may require numerous susceptible cell lines. Even with the shell vial culture method, it is cumbersome for handling large volumes of specimens. A procedure has been developed, which is time and cost-saving and uses specific cell lines in a 96-well microtitre plate and monoclonal antibodies (RETCIF-rapid enhanced tissue culture immunofluorescence). Each clinical sample was inoculated into 12 different wells with five different cell lines. Enhancement was achieved by sonication, centrifugation and hormonal supplementation to the medium used. Cytomegalovirus (CMV), herpes simplex virus (HSV) and respiratory viruses were detected by monoclonal antibodies on day 2, whilst varicella zoster virus (VZV) and enteroviruses were detected on days 5 and 7, respectively. During July-December 1998, 3298 patient specimens were compared by RETCIF and a modified shell vial method. Either or both methods isolated 779 viruses (24% positivity rate), whilst both methods detected 621. Of the 779 viruses, 87% (679) were isolated by the shell vial method in an average time of 4.9 days. For RETCIF the respective rate was 92.5% (721), in an average time of 3.0 days. The RETCIF method is a time-saving procedure, with higher isolation rates than the shell vial method.     

Public Health Laboratory Service enzyme linked immunosorbent assay for detecting Toxoplasma specific IgM antibody.

An enzyme linked immunosorbent assay (ELISA) based on the antibody class capture method for the detection of specific IgM against Toxoplasma gondii, using the microtitre plate format, was developed. Antigen binding was detected using a monoclonal antibody, CIE3, conjugated to horseradish peroxidase. Prior mixing of the conjugate and antigen improved the stability of these reagents as well as removing an incubation stage from the assay. The incubation time of less than four hours permits a rapid throughput of specimens. Using the assay, a total of 163 sera were examined in a three centre study and good agreement was found. Results were expressed as arbitrary enzyme immunoassay units (EIUs) against a freeze dried standard. Throughout the study the standard serum showed a coefficient of variation less than 10% across the microtitre plate. By measuring IgM titres in patients having toxoplasmic lymphadenopathy with a known date of onset, IgM class antibodies were shown to peak at two months, persisting for about six months. In addition, a case of laboratory acquired toxoplasmosis was monitored. Sera shown to contain rheumatoid factor and antinuclear factor did not give false positive results. This rapid, robust, and simplified assay is used by the Public Health Laboratory Service Toxoplasma Reference Units and will provide a standard with which other assays can be compared.     

A sensitive micromethod for measuring human reverse haemolytic plaque-forming cells

A micromethod is described for detecting human immunoglobulin-secreting cells (ISC) using a reverse haemolytic plaque assay (RHPA). Plaques are developed in monolayers of indicator erythrocytes on the botton of the wells in 96-well, flat-bottomed platic microtitre trays. The technique detects more ISC than the conventional RHPA in agarose and this increased sensitivity is not due to the formation of artifact plaques. Together with its relative simplicity and low cost, the sensitivity of the microwell method makes it suitable for use in large scale studies of human B lymphocyte function.     

Primary plate identification of group A beta-hemolytic streptococci utilizing a two-disk technique.

A two-disk system is described which allows primary plate identification of group A beta-hemolytic streptococci. Group A beta-hemolytic streptococci could be visualized on primary throat culture plates by using trimethoprim-sulfamethoxazole to inhibit normal flora. In the heavily inoculated area of Trypticase soy agar plates containing 5% sheep blood, a 25-microgram/ml trimethoprim-sulfamethoxazole disk was placed contiguous to a 0.04-U bacitracin disk. A total of 259 throat specimens were examined with this two-disk system. The swabs from these throat specimens were incubated in Todd-Hewitt broth. The bacterial pellet from the broths was stained by fluorescent antibody as a control. Of the cultures that were determined to be positive on the plates, 75% could be read unequivocally after overnight incubation, whereas the remaining 25% required subculture. The plates recovered 91% of the cultures which were considered as true positives by the broth-fluorescent-antibody technique. This method provided a significant savings in time compared with standard plate methods and in cost of materials compared with broth-fluorescent-antibody methods. This technique is particularly valuable for producing rapid results in laboratories where fluorescence microscopy would not be cost-effective.     

Clinical experience with ultra-rapid freezing of embryos

A modified ultra-rapid freezing technique was introduced in January 1989 in our clinical in-vitro fertilization freezing programme. Thus, we wish to report our 1 1/2 year experience with ultra-rapid freezing as a simple and inexpensive method of routine cryopreservation. Of 181 frozen-thawed embryos, 110 (61%) survived with 50-100% blastomeres intact, and were used in 91 embryo replacements. Nine clinical pregnancies were established including two sets of twins. There was one miscarriage and one ectopic pregnancy. So far, five patients have delivered six normal, healthy infants and two other pregnancies are normally ongoing, including another twin pregnancy. These results were similar to those obtained by the conventional slow freezing method using propanediol. There is no doubt that there is a marked advantage of ultra-rapid freezing when compared to previous methods in terms of cost and time. We therefore believe that more clinical attention should be given to ultra-rapid freezing and it should become increasingly the method of choice for human embryo freezing in IVF programmes.     

Detection of Aspergillus fumigatus PCR products by a microtitre plate based DNA hybridisation assay.

AIMS: To develop a DNA based plate hybridisation assay for the detection of polymerase chain reaction (PCR) products amplified from Aspergillus fumigatus DNA; and to determine the sensitivity of this technique and compare it with Southern blotting. METHODS: A half-log dilution series of DNA extracted from A fumigatus was amplified with specific primers, one of which was 5' end labelled with biotin. PCR products were subsequently detected by agarose gel electrophoresis, Southern blotting, and binding of the products to a streptavidin coated microtitre well, followed by non-radioactive colorimetric detection. Amplification was carried out 10 times for each DNA dilution and a plot of initial DNA concentration against signal intensity was made. RESULTS: A DNA concentration of 1.5 pg could be detected by agarose gel electrophoresis and Southern blotting with a non-radioactively labelled aspergillus specific probe; 1.5 pg was detectable by streptavidin binding of the PCR products to a microtitre plate. The signal from the microtitre plate detection was proportional to the amount of DNA in the PCR reaction on a log-log scale between 100 and 1 pg of DNA. CONCLUSIONS: A DNA based plate hybridisation assay for the detection of A fumigatus PCR products is as sensitive as Southern blotting. However, results are obtained in three hours rather than the three days required for agarose gel electrophoresis, blotting, hybridisation, and detection.