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The immunochemistry of sandwich ELISAs. II. A novel system prevents the denaturation of capture antibodies.

The studies reported here describe a model system by which the effect of surface adsorption on the antigen capture capacity (AgCC) of monoclonal antibodies (MoAbs) can be quantitated. Three mouse anti-fluorescein (FLU) MoAbs were biotinylated and iodinated with [125I]Na. The AgCC/ng of these MoAbs was compared when they were adsorbed directly on plastic or immobilized via a Protein Avidin Biotin Capture (PABC) system. In this system, biotinylated MoAbs were allowed to adsorb on biotinylated carrier proteins through a "bridging" process using Streptavidin. This permits MoAbs to complex with antigen at some distance from the plastic surface. The AgCCs of the MoAbs immobilized using the PABC system were 5-400-fold higher than when they were directly adsorbed on plastic.

Antibodies, Monoclonal↗

Detection of IgG antibodies to measles virus using monoclonal antibodies for antigen-capture in enzyme immunoassay.

An enzyme immunoassay (EIA) for the detection of IgG antibodies to measles viral hemagglutinin (H) has been developed. Monoclonal antibodies were employed as antigen-capture reagents on a polystyrene ball solid phase. The antigen-capture EIA was significantly more sensitive than hemagglutination inhibition (HAI) and somewhat more sensitive than indirect immunofluorescence and indirect EIA for the detection of antibodies to measles virus. The importance of selecting a monoclonal antibody with a high binding affinity, and controlling for non-specific adherence of antigen or antibodies to the solid phase were demonstrated. This method offers not only greater sensitivity than HAI, but also the practicality of reagents capable of standardization and long term storage.

Antibodies, Monoclonal↗

Antigen capture ELISA for the identification of dengue viruses.

We developed a simple antigen capture enzyme immunosorbent assay (AgC-ELISA) for identifying dengue (DEN) virus. The method employed serotype-specific monoclonal antibodies as capture antibodies and an enzyme conjugate of a flavivirus-reactive monoclonal antibody as a detecting antibody. Most DEN virus strains, representing all 4 serotypes isolated from various parts of the tropics, were identical either visually of spectrophotometrically when infected cell culture fluids were used as sources of antigen. The shelf life of the solid phase presensitized with monoclonal antibodies was 4 mth at -15 degrees C. DEN prototype viruses were still identified after storage at -15 degrees C for 1 yr or at room temperature for 1 mth.

Animals↗

Clinical validation of an antibody-capture anti-rubella IgM-ELISA.

An antibody-capture IgM-ELISA using monoclonal antibodies for conjugate was subjected to clinical validation with respect to sensitivity and specificity. In 103 serum specimens, known to contain anti-rubella IgM by a sucrose density gradient method, IgM was found by the ELISA in 99 sera. In a second study, 16 out of 17 acute rubella infections were detected by the IgM-ELISA. In 17 out of 17 vaccinees, a specific IgM response could be demonstrated. Specificity of the antibody-capture ELISA was found to be high; no interference was seen in 60 rheumatoid-factor positive sera, in 100 highly positive IgG sera or 10 sera with anti-CMV IgM. Only one out of 100 sera with heterophile antibodies showed a positive response. In acute rubella infections, IgM was shown to be detectable from 1 to 4 days after onset of illness up to about 12 wk, with peak values at about 1 wk after onset.

Antibodies, Viral↗

The selection and performance of monoclonal and polyclonal anti-respiratory syncytial virus (RS) antibodies in capture ELISAs for antigen detection.

Six monoclonal antibodies directed against fusion protein (F) or nucleoprotein (NP) of respiratory syncytial virus (RS) have been investigated in an antigen capture ELISA for virus detection. The potency, spectrum and pattern of reactivity were investigated with the intention of selecting antibodies reacting with RS-common antigen determinants and with complementary rather than competitive activity. Two anti-F protein antibodies satisfied these criteria and were used with enzyme amplified detection in a two site monoclonal assay (MCA/MCA) or as detectors with a polyclonal antibody as capture (PCA/MCA). Comparative studies were performed with immunofluorescence (FA) as the reference test and nasopharyngeal aspirates processed in different ways. The PCA/MCA assay was superior to that using monoclonal antibodies alone and gave results comparable to the reference method. However, the apparent sensitivity related to FA varied with the type of sample processing used. These results emphasise the need for a critical analysis of the factors which can influence the sensitivity of a particular assay system before judgements on relative sensitivity are made.

Antibodies, Monoclonal↗

A comparison of reverse transcriptase and antigen capture assays for the detection of HIV.

HIV antigen capture assay (ELISA) and reverse transcriptase assay were compared for the detection of HIV. Purified virus and the supernatant of infected cells were used for this study. Antigen capture assay was found to be at least one hundred times more sensitive than the RT assay, and for a large number of samples easier to perform. This assay would be useful for clinical and laboratory diagnosis.

Acquired Immunodeficiency Syndrome↗

Development and evaluation of an IgM capture enzyme immunoassay for diagnosis of recent Norwalk virus infection.

A capture enzyme immunoassay specific for Norwalk IgM class antibody was developed. The assay was moderately sensitive, identifying 33/53 (62%) of patients with naturally acquired Norwalk virus infection and 17/18 (94%) of experimentally infected volunteers. The assay was also specific for IgM class antibody and acute Norwalk virus infection and results were generally reproducible. A specific IgM response correlated with seroconversion by total antibody blocking assay and occurred independently of clinical symptoms. Among 81 symptomatic cases composing seven Norwalk outbreaks, specific IgM was absent from acute phase sera collected less than or equal to three days post onset, and was uncommon in sera collected within one week and after five weeks, with an optimal collection time at about two to three weeks. The Norwalk IgM capture immunoassay may be used to augment paired sera assays in the identification of Norwalk-associated outbreaks of acute gastroenteritis.

Antibodies, Viral↗

IgM antibody capture haemadherence test (MACHAT) for the detection of measles specific IgM.

An antibody capture haemadherence test (MACHAT) for detecting measles-specific IgM is described. The assay is based on the antibody capture principle with rhesus monkey erythrocytes as detector system in place of labelled antisera. MACHAT was compared with a commercial indirect enzyme immunoassay (EIA) for measles-specific IgM using 382 sera from patients notified as measles. There was good agreement between the two tests; 106 sera were found to contain measles IgM by both tests, 7 further sera were positive only in the commercial EIA and 9 only in MACHAT. One sera gave an equivocal result in MACHAT and another in the commercial EIA. Twelve of the 18 sera with discrepant results were also tested by MACRIA; in 7 MACRIA gave the same results as MACHAT, in 3 the MACRIA results agreed with the commercial test and in 2 the MACRIA results were equivocal. Specificity was established by a lack of MACHAT reactivity in sera collected from blood donors (n = 83) and from cases of recent rubella, dengue and parvovirus B19 infection (n = 51). The MACHAT is a simple, cheap test that can be read by eye and is suitable for measles surveillance programmes in the developing world.

Hemadsorption Inhibition Tests↗

Rapid detection of enterovirus in clinical specimens using PCR and microwell capture hybridization assay.

A rapid detection method of enteroviral RNA in clinical samples using PCR and a microwell capture hybridization assay is described. PCR products were labelled directly by digoxigenin-dUTP during the amplification step. The labelled amplicons were hybridized with a biotinylated oligo-probe and captured on commercially available test microwells coated with streptavidin. The hybridized amplicons labelled with digoxigenin were detected using anti-digoxigenin Fab fragments conjugated to peroxidase and colorimetric reaction automatically measured. This method detected as few as 0.01 PFU/100 microl of biological sample with a result obtained within 8 h. Using this method, we were able to detect enteroviral RNA in 23 of 35 clinical specimens from 16 of 17 patients with suspected acute or chronic enteroviral infection. The samples included cerebrospinal fluid, broncho-pulmonary lavage, pericardial effusion, throat swabs, stools, sera, muscular and myocardial biopsies. In contrast, virus was isolated in cell culture in only 8 of 28 clinical specimens from 6 of the 17 patients. This easy-to-perform assay has useful potential in the rapid detection of enterovirus in acute or chronic infection. This methodology could be used for a rapid qualitative detection of other RNA viruses.

Enterovirus↗

Dosimetry and fluence measurements with a new irradiation arrangement for neutron capture therapy of tumours in mice.

Incorporation of 10B in human tumours treated with fast neutrons would increase the local dose in the tumour. In tissue the neutrons are thermalized mainly causing the neutron capture reaction 10B (n,a) 7Li. The dose enhancement can be calculated using the thermal neutron fluence measured by activation of gold foils in a phantom. At a phantom depth of 5 cm a dose enhancement of (0.056 +/- 0.0028)% for each microgram of 10B per gram of tissue was determined in the tumour. A therapeutic gain by this dose enhancement in fast neutron therapy should be examined for a solid tumour in a mouse. The feasibility for neutron capture therapy of tumours in mice depends on the distribution of the tumour dose and on the effective shielding of the bodies of the mice. Therefore, a special holding device for simultaneous irradiation of four tumours was developed. The dose distribution in the tumours and in the surrounding bodies of mice was measured with TLD-300 in a special mouse phantom.

Animals↗

Characterization of the adsorptive pinocytic capture of a polyaspartamide modified by the incorporation of tyramine residues.

Previously it has been shown (Duncan, R., Starling, D., Rypácek, F., Drobník, J. and Lloyd, J.B. (1982) Biochim. Biophys. Acta 717, 248-254) that incorporation of tyramine residues into poly (alpha, beta-(N-2-hydroxyethyl]-DL-aspartamide (PHEA) greatly increases its rate of pinocytic uptake by rat visceral yolk sacs cultured in vitro. Here we describe the relationship between the tyramine content (1.2-21.9 mol%) of modified PHEA and its rate of uptake by yolk sacs. Above a level of substitution of approximately 10 mol% the rate of uptake rises rapidly, and the concentration-dependence of capture is indicative of uptake by adsorptive pinocytosis. Serum proteins were shown to compete effectively for membrane binding sites, indicating a nonspecific interaction of PHEA-derivatives with the yolk sac membrane. PHEA derivatives of the same tyramine content, but of different mean molecular weights (Mr), were captured at the same rates.

Adsorption↗

Pinocytosis and phagocytosis: the effect of size of a particulate substrate on its mode of capture by rat peritoneal macrophages cultured in vitro.

Both phagocytosis (of particles) and pinocytosis (of solutes) occur in macrophages. It is not known, however, whether particles, if they are small enough, can enter by pinocytosis, nor whether there is a minimum size of particle capable of triggering phagocytic uptake. These questions have been investigated by studying, in vitro, the uptake by rat peritoneal macrophages of particles ranging in diameter from 30 nm to 1100 nm. Percoll (30 nm diameter) and polystyrene beads (100, 300, 600, 800 or 1100 nm diameter) were 125I-iodinated and their uptake by macrophages was measured in the absence or presence of metabolic and cytoskeletal inhibitors. Since uptake, expressed as an Endocytic Index (microliter/10(6) cells per h), increased steadily with the duration of incubation and was inhibited by low temperature or metabolic inhibitors, it was concluded that true endocytosis, and not a superficial cell-association, was being measured. Rates of clearance increased with increasing particle diameter. The rate of uptake of Percoll was 10-times, and of 100 nm polystyrene beads 100-times, the rate of fluid-phase pinocytosis, as measured by the uptake of 125I-labelled polyvinylpyrrolidone. Polystyrene beads of 1100 nm diameter were captured at 700-times this rate. The differential effects of colchicine and cytochalasin B on the uptake of 125I-labelled polyvinylpyrrolidone and of 1100 nm polystyrene beads were taken as indicators of their effects on pinocytosis and phagocytosis respectively. It is concluded that Percoll, although particulate, is captured by pinocytosis. The pattern of inhibition of uptake of polystyrene particles suggests that there is no radical discontinuity between pinocytic and phagocytic uptake, but that the contribution of phagocytosis steadily increases with increasing particle diameter. The results are discussed.

2,4-Dinitrophenol↗

Boron neutron capture enhancement of 252Cf brachytherapy.

Dosimetric and radiobiological studies were undertaken to investigate the potential enhancement in dose, dose distribution and cell killing effectiveness of 252Cf brachytherapy achievable when boron-10 enriched compounds are incorporated into simulated 252Cf brain implants. Thermal neutron distributions in a human head phantom containing a 252Cf source were measured by gold foil activation and calculated using a 1-dimensional transport code. This information was then used to modify measured event size distributions for 252Cf neutrons to determine the corresponding increase in dose and dose equivalent throughout the phantom. The addition of subtoxic levels of boron-10 to a typical 252Cf implant was found to significantly enhance both the absorbed dose and the high LET event frequency at distances of 3 to 5 cm from individual sources. Some unexpected geometric considerations are discussed. Reduced survival of cultured Chinese hamster cells correlated with the predicted increase in absorbed dose from the capture events with a concentration of about 60 micrograms 10B per ml in the culture medium. It was found that boron increased alpha (the "single-hit" parameter of the linear quadratic survival model) by 32% and decreased beta (the "double-hit" parameter) by 8%. The alpha/beta ratio increased to 4.34 Gy in the presence of boron, from 3.03 Gy in its absence. This translated to an 8% reduction in californium dose needed to effect 10% cell survival. It is concluded that there is a sufficiently high thermal neutron fluence present during californium brachytherapy for boron neutron capture dose augmentation to be feasible.

Animals↗

Detection of hog cholera virus antigens in experimentally-infected pigs using an antigen-capture ELISA.

An antigen-capture ELISA was used to detect hog cholera virus (HCV) antigens in blood and tissues taken from pigs infected with 2 different strains of virus. Specific antigens were demonstrated in peripheral blood leucocytes (PBLs) and a wide range of tissue samples 4-6 days after infection of pigs with a moderate-high virulent HCV strain (Weybridge virus). Strong signal to noise (S/N) ratios were obtained in the ELISA for PBLs and lymphoid tissues such as spleen, tonsil and mesenteric lymph nodes at 5-7 days after infection with the Weybridge virus, S/N ratios varying between 8.1-19.7 for blood samples and 4.3-19.1 for spleen samples. High positive ELISA results were also obtained for duodenum and ileum samples (S/N ratios 10.3-18.6) taken from these pigs, reflecting severe pathological changes observed in the gut at post mortem. In contrast, the antigen-capture ELISA gave strong positive results for PBLs and spleen samples only at 7-9 days after infection of pigs with a low virulent strain of HCV (New South Wales virus). The ELISA S/N ratios averaged 9.5 for PBLs and 8.9 for spleen samples in these animals. Although virus isolation detected infection earlier in the infected pigs, the ELISA returned positive results on PBLs and spleen samples around the time all of the animals first showed typical signs of classical swine fever. The technique does not require tissue culture and takes less than 36 h to return a definitive result.

Animals↗

Determination of endotoxins by gas chromatography: evaluation of electron-capture and negative-ion chemical-ionization mass spectrometric detection of halogenated derivatives of beta-hydroxymyristic acid.

The sensitivity and selectivity of gas chromatography for analysing several halogenated ester derivatives of beta-hydroxymyristic acid were studied using both selected-ion monitoring detection with negative-ion chemical-ionization mass spectrometry and electron-capture detection. Six different derivatization methods were compared with respect to yield, chemical stability and formation of by-products. Procedures for removal of excess reagents using disposable silica columns and thin-layer chromatography were elaborated. The 3-O-pentafluorobenzoyl-methyl ester was the preferred derivative since it provided high sensitivity and had the molecular ion as the base peak in the mass spectrum. The detection limit was 0.5 pg with electron-capture detection and 0.3 pg with the mass spectrometric system. Using beta-hydroxymyristic acid as a chemical marker it was possible to detect Escherichia coli endotoxin in aqueous solution at a level of 1 ng/ml.

Chromatography, Gas↗

Capillary gas chromatographic-electron-capture assay for the aldose reductase inhibitor imirestat in lens and plasma.

A sensitive and selective gas chromatographic-electron-capture assay was developed for the determination of the aldose reductase inhibitor imirestat in lens and plasma. The method involves solid-phase extraction of drug and internal standard from the plasma specimen or lens sample homogenate using "Baker"-10 SPE extraction columns followed by derivatization with pentafluorobenzyl bromide and further purification. Derivatives of drug and internal standard were separated on a fused-silica capillary column and analyzed using a 63Ni electron-capture detector. The limit of detection was 2.5 ng per lens or ml of plasma. The method was used to evaluate the pharmacokinetics of imirestat in human subjects and to quantitate imirestat in animal lens tissue following topical ocular administration.

Administration, Topical↗

Analysis of pipecolic acid in biological fluids using capillary gas chromatography with electron-capture detection and [2H11]pipecolic acid as internal standard.

A sensitive and accurate stable isotope dilution assay was developed for the measurement of pipecolic acid in body fluids using capillary gas chromatography with electron-capture detection. The method utilizes [2H11]pipecolic acid as the internal standard. Sample preparation consisted of derivatization in aqueous solution (pH 11.5) of the amine moiety with methyl chloroformate to the N-methylcarbamate, followed by acidic ethyl acetate extraction at pH less than or equal to 2 and further derivatization of the carboxyl moiety with pentafluorobenzyl bromide, the excess of which was removed by solid-phase extraction. Control values have been determined in the plasma of at-term infants, age greater than 1 week (n = 21, mean = 1.36 microM, range = 0.47-3.27 microM). The utility of the method was demonstrated by quantitating pipecolic acid in biological fluids derived from patients with peroxisomal disorders. The method was validated against an established electron-capture negative ion mass fragmentographic technique.

Body Fluids↗

Packed-column supercritical fluid chromatography of artemisinin (qinghaosu) with electron-capture detection.

In this preliminary report, a supercritical fluid chromatographic method is described for the determination of artemisinin in whole blood. The chromatography is carried out on a 20 cm x 1 mm I.D. Deltabond cyano supercritical fluid chromatographic column with detection of the artemisinin via an electron-capture detector. The sample work-up uses a liquid-liquid extraction with hexane, giving a recovery of 82%. The current limit of detection using 1 ml of blood is 20 ng/ml. We speculate that the endoperoxide moiety accounts for the response to the electron-capture detector and thus provides a new approach by which this class of compounds may be analyzed.

Antimalarials↗