Resolution of dimly fluorescent particles: a practical measure of fluorescence sensitivity.

Flow cytometry is usually used to analyze subpopulations of cells, not simply to measure the mean fluorescence level of a mixture. Thus, resolution or coefficient of variation (CV) of dimly stained populations is the most appropriate measure of fluorescence "sensitivity." Methods used to measure sensitivity that are in routine use do not unambiguously and completely determine the ability of a flow cytometer to resolve dimly fluorescent populations from each other. Since fluorescence sensitivity depends on two factors, background light (B) and detection efficiency (Q, the detected photoelectrons per fluorochrome molecule on an analyzed particle), one cannot uniquely define the operating condition of a flow cytometer with just one of these factors. In general, it is not possible to define the ability of a flow cytometer to resolve dim subpopulations by using a single number such as "noise level" or "detection threshold"-the description requires a "two-parameter" measure. A carefully characterized flow cytometer was used to determine the inherent fluorescent CV of dimly fluorescing beads. The fluorescence from the beads is also calibrated in terms of molecules of equivalent soluble fluorophore (MESF). The beads with known inherent CV and MESF provide a standard against which the instrument contribution to the CV of dim fluorescence can be measured. By measuring the standard deviation (SD) of the fluorescence histogram from unstained beads (noise) we obtain a second measure of instrument performance. The bead CV and noise SD are a sufficient pair of factors to determine the optical capability of a flow cytometer to resolve dim subpopulations of particles. It is also possible to use the measurements to calculate B and Q and use this information to predict the shapes of fluorescence histogram distributions of dim particles.

The sensitive detection and quantitation of antibody to HCV by using a microsphere-based immunoassay and flow cytometry.

Antibody to HCV core and NS3 was quantified by using a microsphere immunoassay and flow cytometry. Antibody to core and NS3 was elevated in the 85 seropositive blood donors tested. The amount of either antibody varied over two logs although greater variation was seen with the antibody to NS3 than was seen with antibody to core. In three documented acute HCV cases, the microsphere assay detected antibody prior to antibody detection using the reference methods. Twenty donor samples were indeterminate by the reference methods: 45% of these were indeterminate with the microsphere assay while 25% were negative and 30% were positive. As compared to enzyme immunoassay the microsphere assay showed a 5-fold increase in sensitivity. The microsphere assay demonstrated increased sensitivity for the quantification of specific antibody to HCV core and NS3 and was useful in resolving a significant proportion of indeterminate samples.

Neutralizing antibody to human rhinovirus 14 penetrates the receptor-binding canyon.

The three-dimensional structure of intact human rhinovirus 14 (HRV-14) complexed with Fab fragments (Fab17-IA) from a strongly neutralizing antibody that binds bivalently to the virion has been determined to 4.0 angstrom resolution by a combination of X-ray crystallography and cryo-electron microscopy. In contradiction to the most commonly held model of antibody-mediated neutralization, Fab17-IA does not induce a conformational change in the HRV-14 capsid. Instead, the paratope of the antibody undergoes a large conformational change to accommodate the epitope. Unlike any previously described antibody-antigen structure, the conserved framework region of the antibody makes extensive contact with the viral surface. Fab17-IA penetrates deep within the canyon in which the cellular receptor for HRV-14 binds. Hence, it is unlikely that viral quaternary structure evolves merely to evade immune recognition. Instead, the shape and position of the receptor-binding region on a virus probably dictates receptor binding and subsequent uncoating events and has little or no influence on concealing the virus from the immune system.

Structure of a human rhinovirus-bivalently bound antibody complex: implications for viral neutralization and antibody flexibility.

The structure of a neutralizing immunoglobulin (monoclonal antibody mAb17-IA), bound to human rhinovirus 14 (HRV14), has been determined by cryo-electron microscopy and image reconstruction. The antibody bound bivalently across icosahedral twofold axes of the virus, and there were no detectable conformational changes in the capsid. Thus, bivalently bound IgGs do not appear to cause gross deformations in the capsid. Differences between the electron density of the constant domains of the bound Fab fragment and IgG structures suggested that conformational changes occur about elbow axes upon bivalent attachment as was previously predicted. No significant density was observed for the Fc fragment, which adds further evidence for a high degree of mobility about the hinge region.

Structure of human rhinovirus complexed with Fab fragments from a neutralizing antibody.

We have determined the structure of a human rhinovirus (HRV)-Fab complex by using cryoelectron microscopy and image reconstruction techniques. This is the first view of an intact human virus complexed with a monoclonal Fab (Fab17-IA) for which both atomic structures are known. The surface area on HRV type 14 (HRV14) in contact with Fab17-IA was approximately 500 A2 (5 nm2), which is much larger than the area that constitutes the NIm-IA epitope (on viral protein VP1) defined by natural escape mutants. From modeling studies and electrostatic potential calculations, charged residues outside the neutralizing immunogenic site IA (NIm-IA) were also predicted to be involved in antibody recognition. These predictions were confirmed by site-specific mutations and analysis of the Fab17-IA-HRV14 complex, along with knowledge of the crystallographic structures of HRV14 and Fab17-IA. The bound Fab17-IA reaches across a surface depression (the canyon) and meets a related Fab at the nearest icosahedral twofold axis. By adjusting the elbow angles of the bound Fab fragments from 162 degrees to 198 degrees, an intact antibody molecule can be easily modeled. This, along with aggregation and binding stoichiometry results, supports the earlier proposal that this antibody binds bivalently to the surface of HRV14 across icosahedral twofold axes. One prediction of this model, that the intact canyon-spanning immunoglobulin G molecule would block attachment of the virus to HeLa cells, was confirmed experimentally.

Purification and crystallization of intact human rhinovirus complexed with a neutralizing Fab.

We report the first crystallization of an intact virion, human rhinovirus 14, complexed with the Fab fragment from a neutralizing antibody. These crystals diffract to at least 6.0A resolution. It has been suggested that Fab's and mAb's can induce large conformational changes in the capsid upon binding. The structure of this complex should enable us to detect the existence and role of such changes.

Detection of the human erythrocyte surface antigen gerbich by flow cytometry using human antibodies and phycoerythrin for extreme immunofluorescence sensitivity.

We used indirect immunofluorescence and flow cytometry to detect the human erythrocyte surface antigen Gerbich. The procedure consisted of sequential erythrocyte-labeling with human antibody to Gerbich, biotinylated goat anti-human IgG and finally phycoerythrin-conjugated streptavidin. With maximal excitation of phycoerythrin at 546 nm, an increase in the fluorescence sensitivity was achieved. All erythrocytes from normal controls had detectable Gerbich antigen with little variation in antigen density between individuals. Six Gerbich-negative patients had no detectable antigen. By diluting the erythrocytes, as few as 0.1% antigen-positive cells in an antigen-negative population could be detected. These studies indicate that flow cytometry is a useful technique for the detection of erythrocyte surface antigens.

Trichotillometry: the quantitation of hair pluckability as a method of nutritional assessment.

In protein-calorie malnutrition, particularly kwashiorkor, ease of hair pluckability is frequently observed. In an attempt to quantitate this manifestation of the disease a calibrated mechanical instrument, referred to as a trichotillometer, was devised and used to determine the force required to epilate individual hairs in 17 adult hospitalized patients with evidence of protein calorie malnutrition and in 16 well-nourished patients. Nutritional status was examined by measuring body weight, triceps skinfold thickness, arm muscle circumference, serum albumin, lymphocyte count, hematocrit, beta-carotene, vitamin nutriture, and hair shaft diameter. Average epilation force was significantly lower in the malnourished patients than in the well nourished group (17.0 g +/- 11.8 versus 38.2 g +/- 11.4, p less than 0.001), with the lowest mean value found in patients categorized as having kwashiorkor (14.8 g). Plucking force correlated significantly and positively with serum albumin, hair shaft diameter, triceps skinfold, arm muscle circumference, weight, hematocrit, beta-carotene; it did not correlate with vitamin status. In order to determine the effect of acute stress on epilation force a subgroup of 18 patients was evaluated before and 1 and 4 days after surgery. Within this time interval epilation force was not significantly altered by the stress of surgery.

DNA damage in rat 9L cells treated with nitrogen mustard and 1,3-bis(2-chloroethyl)-1-nitrosourea assayed by viscoelastometry and S1 nuclease.

The techniques of viscoelastometry and S1 nuclease digestion were applied to the analysis of DNA damage in rat 9L cells treated with nitrogen mustard and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Results of the S1 nuclease assay permitted quantitation of the amount of single-strand (or alkali-labile) break formation as well as DNA interstrand cross-link formation. In the presence of 2% detergent, only cells treated with nitrogen mustard showed evidence of DNA cross-link formation as determined by this assay. Viscoelastic analysis of cell lysates under denaturing conditions (pH 12.15) showed that cells treated with nitrogen mustard led to substantial increases in both the viscoelastic retardation time and recoil, consistent with the presence of DNA cross-links, while treatment with BCNU led to decreases in these two properties, consistent with the induction of single-strand breaks. Viscoelastic analysis of cell lysates under nondenaturing conditions (pH 11.15) showed that nitrogen mustard produced an increase in retardation time, consistent with single-strand break induction, along with a fast recoiling component that eventually led to gel-like behavior, suggesting the possibility of drug-induced intermolecular DNA-DNA cross-links. BCNU treatment resulted in a decrease in retardation time. This decrease in consistent with induction of DNA interstrand cross-links by BCNU and shows that the single-strand breaks observed at denaturing conditions were due to the presence of alkali-labile sites rather than true strand breaks. While other methods using denaturing conditions have resulted in evidence for DNA cross-links following BCNU treatment, both viscoelastic and S1 nuclease experiments showed negative results in this regard. Further work is needed to clarify this point.

Viscoelastic behavior of mammalian DNA.

The viscoelastic behavior of rat 9L cellular DNA was studied as a function of the detergent used for lysis, the pH and duration of lysis, and gamma ray dose. For nondenaturing lysis conditions, a model of the DNA was proposed to account for the effects of these agents on the viscoelastic retardation time. It was concluded that these agents affect the hydrodynamic radius of the DNA rather than its molecular weight. For denaturing lysis conditions, molecular weights calculated from the relaxation time were consistent with those calculated from alkaline sucrose sedimentation profiles.

Home birth in Salt Lake County, Utah.

In Salt Lake County, Utah in 1972 and 1975, 167 women had planned home deliveries. Birth certificate data indicated they did not differ from the total population in age, marital, and socioeconomic status. Eighty-four women were interviewed and reported hostility from health professionals which may have placed them at unnecessary risk.