Detection of rotavirus with a new polyclonal antibody enzyme immunoassay (Rotazyme II) and a commercial latex agglutination test (Rotalex): comparison with a monoclonal antibody enzyme immunoassay.

A total of 176 human fecal specimens were examined for the presence of rotavirus by four different assays: a monoclonal antibody enzyme immunoassay; the original polyclonal antibody enzyme immunoassay marketed by Abbott Laboratories, North Chicago, Ill. (Rotazyme I); a modification of this assay which is now commercially available (Rotazyme II); and a latex agglutination test (Rotalex) recently introduced by Medical Technology Corp., Somerset, N.J. In addition, selected specimens were examined for the presence of rotavirus by electron microscopy, immune electron microscopy, and RNA gel electrophoresis. A total of 40 specimens were positive in the monoclonal antibody enzyme immunoassay, and 136 were negative. Using the results obtained with this procedure as the reference standard, we found the sensitivities of the Rotazyme I, Rotazyme II, and Rotalex tests to be 97.4, 100, and 81.6%, respectively. The specificities of these three procedures were 88.8, 83.9, and 100%, respectively.     

Confirmation of positive antibody screens by solid-phase red cell adherence assay using a tube technique method with polyethylene glycol enhancement

Our blood bank routinely screens donors for antibodies using a solid-phase red cell adherence (SPRCA) assay. Positive results are then confirmed using a tube technique with polyethylene glycol (PEG) enhancement due to reported higher specificity than with SPRCA. Over a 5-month period, 49,084 donor serum or plasma samples were tested using the SPRCA assay. Further identification of positive samples was performed using a PEG enhancement method. Testing was performed with strict adherence to the manufacturers' inserts. Of 49,084 samples, 313 (0.64%) were positive by the SPRCA assay. Of these, 99 (31.6%) samples remained positive when tested with PEG enhancement. The remaining 214 (68.4%) were negative, giving specificity for the SPRCA assay of 99.6 percent (48,985/ 49,199). We report a high specificity for antibody screening using the SPRCA assay. However, it is cost effective to perform a confirmatory tube test with PEG enhancement because 214 SPRCA assay samples were interpreted as having a negative antibody screen, thus allowing the release of valuable blood components for transfusion.     

A comparison of a new affinity column system with a conventional tube LISS-antiglobulin test for antibody detection

A recently introduced system for antibody detection (ReACT) consists of affinity columns (AFC) that contain protein A and protein G-coated agarose. We compared the ReACT system to a conventional tube low-ionic-strength saline antiglobulin test (LISS-AGT). We selected 100 LISS-AGT positive samples with clinically important and benign antibodies of varying strengths and 130 LISS-AGT negative samples to evaluate by the AFC method. AFC tests were positive with all 84 clinically important antibodies, including 36 antibodies that reacted <= 1+ at LISS-AGT (0% falsely negative). Eleven of 16 (69%) clinically benign antibodies reacted by AFC. Five samples (2 anti-Sda, 2 anti-I, and 1 inconclusive) were negative by AFC. AFC tests were negative with all 130 samples that were negative by LISS-AGT (0% falsely positive). The AFC method showed results comparable with results obtained with a conventional tube LISS-AGT for detection of clinically important antibodies. Some unwanted, clinically benign antibodies may not be detected by the AFC method.     

A double antibody radioimmunoassay for mouse hemoglobins: use of polyethylene glycol in conjunction with the second antibody.

A double antibody radioimmunoassay (RIA) system is described for detection of small quantities of hemoglobins. Mouse (C57BL/6) hemoglobin and horse anti-mouse hemoglobin antiserum were used to develop the system.The first phase of the RIA, i.e., the initial reaction between the antigen and the antibody, was found to be complete within 24 h. The reaction proceeded better at 4°C than at 25°C. The second phase, i.e., separation of bound from unbound antigen, was achieved by precipitation with a second antibody (goat anti-horse IgG) and polyethylene glycol (PEG). A 50 g/l concentration of PEG was found to be best suited for the assay. Mixing of all the reagents together was found to decrease the binding as compared to the system in which second antibody and PEG were added after completion of the first phase. Maximum precipitation of the complex took place within 30 min after the addition of the second antibody and 1 h after the addition of 50 g/l PEG.The RIA system described here combines the conventional double antibody RIA with the PEG method. This method has decreased the amount of time necessary for precipitation from 24 h (or longer) to 1 h. Large molecular weight antigens could not be estimated in the conventional PEG method because of their insolubility in 200 g/l PEG utilized in the assay. The use of a low concentration of PEG along with the second antibody in the method described here allows the estimation of large molecular weight antigens. This double antibody-PEG method has a general applicability for small as well as large molecular weight antigens.     

Cell biotinylation provides a sensitive and effective detection technique for cellular adhesion assays: comparison with existing methods

A simple, sensitive, colorimetric labelling method was devised to quantify cell adhesion, based on labelling the cell plasma membrane with biotin. This method was applied in adhesion assays, which involved the adherence of biotin-labelled, PMA-stimulated, U937 cells. These cells resemble monocytes, and were bound onto fibronectin-coated wells and to an ECV304 cell monolayer. The adherent U937 cells were detected by the addition of a peroxidase-conjugated anti-biotin antibody and a soluble colorimetric substrate. This assay is convenient, fast and sensitive, and able to detect 320-1000 U937 cells under the conditions described.This study has used titration assays to compare the biotinylation method with the existing cell quantification approaches of 51Cr radiolabelling and antibody dependent ELISA. Chromium labelling was the most sensitive technique, but we found the biotinylation method to be more convenient than radioactive labelling and more sensitive than conventional ELISA.Biotinylated cells were also used very effectively in a Stamper-Woodruff adhesion assay with U937 cells binding to histological sections of atherosclerotic plaques. The selective detection of the bound cells permitted automated quantitation by image analysis.Whole cell biotinylation may have wider applications in biological research.     

Limitations of the radioimmunoprecipitation polyethylene glycol assay (RIPEGA) for detection of filarial antigens in serum

The performance of the radioimmunoprecipitation polyethylene glycol assay (RIPEGA) was examined for quantitation of filarial antigens (Brugia malayi and Dirofilaria immitis) in serum from infected human and animal hosts and non-infected controls. Multiple PEG concentrations were employed to determine the level of non-specific binding (NSB) in non-exposed human sera (NEHS) containing no filarial antigen. The NSB observed when 3 different 125I-labeled IgG antibodies were added to 26 NEHS varied 3-fold and was correlated significantly with total serum IgM (r = 0.80, P less than 0.005, n = 24) but not with serum IgA (r = 0.37) or IgG (r = 0.45). NSB levels were significantly reduced when a Fab'2 fragment of the 125I-labeled antibody was used, but the correlation of NSB with total serum IgM remained significant (r = 0.57, P less than 0.01). The presence of rheumatoid factor in NEHS sera also significantly increased NSB by an average of 3-fold. These effects eliminated the assay's ability to detect in sera from infected hosts filarial antigen the presence of which could be readily demonstrated by an immunoradiometric assay. The RIPEGA's precision (intra-assay coefficient of variation (CV) = 21% at 35% Bmax) and reproducibility (inter-assay CV = 29% at 35% Bmax) are less satisfactory than many alternative immunoassays. In many cases, positive sera failed to dilute out in parallel with each other or with an antigen-spiked standard reference curve. We conclude that poor performance characteristics currently limit the utility of the RIPEGA for quantitating filarial antigen in human and animal serum.     

Three polyethylene glycol dependent methods for the detection of circulating immune complexes in pathological sera: Comparison with the Raji cell method

A composite method using polyethylene glycol (PEG) and different markers for detecting circulating immune complexes (CIC) is described. The markers used are bovine conglutinin (RK-BA), C1q (C1q-BA) and IgG, IgM quantitation of PEG precipitate (RID-Ig). A composite scoring system is used in interpreting results from individual assays. The sensitivity of multiple PEG methods (MPM) was determined in 418 serum samples and compared with Raji cell assay in 204. Correlations between individual assays, viz., RK-BA, C1q-BA, RID-Ig and Raji cell test in several disease conditions including rheumatoid arthritis, glomerulonephritis, post-renal transplantation, maintenance haemodialysis, multiple sclerosis and normal pregnancies were computed. The relative discriminatory ability of a single PEG technique to differentiate normal from pathological sera in these disease states was observed in comparison with the composite PEG index. This index gives an improved assessment of abnormal sera, is simple and sensitive and has some advantages over biological techniques such as the Raji cell assay.     

ELISA for toxoplasma antibody detection: a comparison with other serodiagnostic tests.

An ELISA method was developed for the measurement of toxoplasma IgG antibodies in human serum using antigen-coated polystyrene beads as a solid phase and anti human IgG-horse radish peroxidase conjugate as an enzymatic tracer. In order to assess ELISA sensitivity and specificity, a between methods comparison was made using 'conventional' serological tests as reference (dye-test, crossover-linked immunoassay, passive haemagglutination, indirect immunofluorescence). From an analysis of the group classifications obtained some considerations emerged: the ELISA specificity looks comparable with that of the 'reference' tests, as no sample classified as negative by all these tests was ELISA-positive, and vice versa; ELISA appears to correlate better with haemagglutination and immunofluorescence, on the basis of the respective class frequencies; in particular, the number of positives, which is much lower for the dye-test and crossover-linked immunoassay, suggests that a higher sensitivity is reached in the former cases.     

Improved detection of weak, clinically significant antibodies by supplementation of polyethylene glycol with a low-ionic solution

A comparative study of 164 serum samples was carried out to determine the specificity and sensitivity of the indirect antiglobulin test (IAGT) in three different formulations: physiologic saline, low-ionic solution (RAM), and RAM supplemented with polyethylene glycol (PEG). Serum samples containing mostly weak antibodies (anti-D, -C, -E, -c, -Jka, -Fya, -K, -S, -Lea, -Lua, -M, -Cob, -P1, -I, and -Kna) were used in a 10-minute IAGT in which PEG-IAGTs were compared with saline- IAGTs and RAM-IAGTs. With the exception of anti-P1, anti-I, and an anti-Lea, PEG-IAGTs detected all the antibodies tested compared with 72.3% and 77.4% for saline-IAGTs and RAM-IAGTs, respectively. The end-point titers of at least 82% of antibodies detected by PEG-IAGTs were 1-3 dilutions higher than those by saline- and RAM-IAGTs. When specificity of PEG-IAGTs was tested using 268 randomly selected, fresh (< 1 day old) blood samples, PEG-IAGT detected 11 out of 268 samples as positive compared with 7 out of 268 by both saline-IAGTs and RAM-IAGTs. The four antibodies that were not detected were identified as anti-D, anti-E, anti-Bga, and an autoantibody known previously to be only reactive with papain-pretreated red cells. No nonspecific reactions were detected by PEG-IAGTs and no hemolysis was evident in any of the IAGTs. PEG-IAGTs were more sensitive than saline- and RAM-IAGTs. PEG-IAGTs detected all weak, clinically significant antibodies as well as four antibodies that were otherwise undetected by saline-IAGT or RAM-IAGT. The overall sensitivity of the PEGIAGT was 96.3% compared with 84.1% and 73.2% for the RAM- and saline-IAGT, respectively.     

A novel low-friction surface for biomedical applications: modification of poly(dimethylsiloxane) (PDMS) with polyethylene glycol(PEG)-DOPA-lysine

Aqueous biocompatible tribosystems are desirable for a variety of tissue-contacting medical devices. L-3,4-dihydroxyphenylalanine (DOPA) and lysine (K) peptide mimics of mussel adhesive proteins strongly interact with surfaces and may be useful for surface attachment of lubricating polymers in tribosystems. Here, we describe a significant improvement in lubrication properties of poly (dimethylsiloxane) (PDMS) surfaces when modified with PEG-DOPA-K. Surfaces were characterized by optical and atomic force microscopy, contact angle, PM-IRRAS, and X-ray photoelectron spectroscopy. Such surfaces, tested over the course of 200 rotations ( approximately 8 m in length), maintained an extremely low friction coefficient (mu) (0.03 +/- 0.00) compared to bare PDMS (0.98 +/- 0.02). These results indicate the potential applications of PEG-DOPA-K for the modification of device surfaces. Extremely low mu values were maintained over relatively long length scales and a range of sliding speeds without the need for substrate pre-activation and in the absence of excess polymer in aqueous solution. These results were only obtained when DOPA was bound to lysine (modification with PEG-DOPA did not have an effect on mu) suggesting the critical role of lysine in obtaining a lowered friction coefficient.     

Development of a simple latex agglutination assay for detection of shiga toxin-producing Escherichia coli (STEC) by using polyclonal antibody against STEC

Rabbit antiserum raised against the whole-cell antigen of Shiga toxin-producing Escherichia coli (STEC) strain VT3 (stx1+ stx2+ eae+) was repeatedly adsorbed with heat-killed cells of different non-STEC strains and other enteric bacteria. Thus, the antiserum obtained was designated VT3 antiserum. VT3 antiserum reacted with intimin type gamma. We assessed the reactivity of VT3 antiserum to whole-cell lysates of 87 strains of E. coli and other enteric bacteria by immunoblotting. The antiserum recognized the 97-kDa protein in whole-cell lysate from strain VT3, and 36 (83.7%) of the 43 STEC strains were positive for the STEC antigen. None of the non-STEC strains or strains of other species examined tested positive by immunoblotting. Based on this result, we developed a latex agglutination assay for the detection of STEC strains. Thirty-five (81.4%) of the 43 STEC strains tested positive for the STEC antigen by the latex agglutination assay. One (3.3%) of the 30 non-STEC strains and none of the strains of the other enteric bacteria included in this study tested positive by the latex agglutination assay. The corresponding specificity of the latex agglutination assay was approximately 98%. Results of this study showed the production of STEC antiserum and the generation of a simple, cost-effective, sensitive, and specific latex agglutination assay for establishing an etiological diagnosis of STEC.     

Mycoplasma detection in cell cultures: a comparison of four methods

Mycoplasma is a common contaminant of tissue culture samples. Infection is persistent, difficult to detect and diagnose, and very difficult to cure. The concentration of mycoplasma in infected cultures can be as high as 10(7) colony-forming units per mL, and their presence can change many of the cell reactions, including altering cell growth rate, inducing morphological changes or cell transformation, and mimicking virus infection. Therefore, it should be assumed that a mycoplasma-contaminated cell line may be significantly influenced in every respect, and, thus, experimental data derived from such a cell line is likely to be invalid. Contamination is not obvious, either macroscopically or microscopically; thus, routine mycoplasma testing is essential for any cell culture laboratory. Many of the testing procedures developed so far are time-consuming, expensive, inconclusive and unsuitable for screening large numbers of test specimens. This study compares DNA staining, enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) and PCR ELISA, to determine which is the best procedure for routine assessment of cell cultures. All four methods gave reproducible results with both infected and non-infected cell lines. Both ELISA methods were easy to perform, reproducible and easily interpreted.     

Faecal occult blood testing: comparison of a latex agglutination test (Hemolex) and an immunoturbidimetric test (QuikRead FOB)

Occult blood detection is the most prescribed faecal examination. AIM: To compare results obtained with the latex agglutination test Hémolex LA (Orion diagnostica, Finlande) with those given by an immuno-turbidimetric test which allows an automatic reading (QuikRead FOB, Orion diagnostica, Finlande). MATERIAL AND METHODS: this prospective study was carried out in 140 patients. The reference method was the latex agglutination test, Hemolex LA performed on stool extract obtained through weighting samples. On the base of the results, samples were separated into 2 groups: positive (n = 45) and negative (n = 95). As the QuikRead FOB test indicated a stool extract obtained through a sampling set, such an extraction was performed before Hemolex LA et QuikRead FOB testing. RESULTS: all the 95 samples from the negative group gave similar results with the 3 methods. In contrast, 12/45 of the positive samples gave conflicting results, 11 results were negative with the 2 tests performed on stool extract obtained via sampling set, 1 result was negative with the QuikRead FOB method only. DISCUSSION: analytical performance were similar with the 2 methods and discrepancies observed wi-thin the positive group were mainly related to the extraction method.     

Comparison of a latex agglutination test with five other methods for determining the presence of antibody against cytomegalovirus.

A latex agglutination test for determination of antibody against cytomegalovirus was compared with five other methods: a solid-phase fluorescent immunoassay, an indirect hemagglutination test, two solid-phase enzyme immunoassays, and an indirect fluorescent-antibody method, with sera collected from 210 random blood donors. Of the sera tested, 28% were positive for anti-cytomegalovirus by concordance of four or more methods. The latex agglutination test performed well, with a sensitivity of 100%, a specificity of 99%, and positive and negative predictive values of 97 and 100%, respectively. The methods were also evaluated for the number of sera requiring repeat testing, equivocal results after retesting, ease of performance, turnaround time, and technical demands. The tests which best met the requirements for a screening test were the solid-phase fluorescent immunoassay, the indirect hemagglutination test, and the latex agglutination test. The latex agglutination test is a valuable screening tool for detecting total anti-cytomegalovirus which has high sensitivity, high negative predictive value, and rare equivocal results and also has the added advantages of ease of performance and rapid turnaround time.