Background:The diagnosis and management of acute ischemic stroke are limited by the lack of rapid diagnostic assays for use in an emergency setting. Computed tomography (CT) scanning is used to diagnose hemorrhagic stroke but is relatively ineffective (<33% sensitive) in detecting ischemic stroke. The ability to correlate blood-borne protein biomarkers with stroke phenotypes would aid in the development of such rapid tests. Methods: ELISAs for >50 protein biomarkers were developed for use on a high-throughput robotic workstation. These assays were used to screen plasma samples from 214 healthy donors and 223 patients diagnosed with stroke, including 82 patients diagnosed with acute ischemic stroke. Marker assay values were first compared by univariate analysis, and then the top markers were subjected to multivariate analysis to derive a marker panel algorithm for the prediction of stroke. Results: The top markers from this analysis were S-100b (a marker of astrocytic activation), B-type neurotrophic growth factor, von Willebrand factor, matrix metalloproteinase-9, and monocyte chemotactic protein-1. In a panel algorithm in which three or more marker values above their respective cutoffs were scored as positive, these five markers provided a sensitivity of 92% at 93% specificity for ischemic stroke samples taken within 6 h from symptom onset. Conclusion: A marker panel approach to the diagnosis of stroke may provide a useful adjunct to CT scanning in the emergency setting.

The location of the cytochrome binding site on the reaction center of Rhodopseudomonas sphaeroides was studied by two different approaches. In one, cross-linking agents, principally dithiobis(propionimidate) and dimethyl suberimidate, were used to link cytochrome c and cytochrome c2 to reaction centers; in the other, the inhibition of electron transfer by antibodies against the subunits was investigated. Cytochrome c (horse) cross-linked to the L and M subunits, whereas cytochrome c2 (R. sphaeroides) cross-linked only to the L subunit. The cross-linked reaction center-cytochrome complexes were isolated by affinity chromatography. The rate of electron transfer in the cross-linked cytochrome c2 complex was the same as that in the un-cross-linked complex. However, when cytochrome c was used, the rate in the cross-linked complex was about 15 times slower than that in the un-cross-linked complex. Fab fragments of antibodies specific against the L and M subunits blocked electron transfer from both cytochrome c (horse) and cytochrome c2 (R. sphaeroides). Antibodies specific for the H subunit did not block either reaction. We conclude that the cytochrome binding site on the reaction center is close (approximately 10 A) to both the L and M subunits, possibly in a cleft between them.

The localization of the reaction center polypeptides (L, M, and H) in the membranes of both the wild-type, strain 2.4.1, and the carotenoidless mutant, R-26, of Rhodopseudomonas sphaeroides was determined by using affinity-purified antibodies specific for these proteins.Binding of the antibodies to reaction center subunits in spheroplasts was visualized in the electron microscope by immunoferritin labeling. The H and M subunits were labeled at both the cytoplasmic and the periplasmic surfaces of the membrane, whereas the,L subunit was labeled only at the periplasmic surface of the membrane. Thus, the reaction center is asymmetrically oriented in the membrane with at least two subunits (H and M) spanning the membrane.The plasma membrane of the photosynthetic bacterium, Rhodopseudomonas sphaeroides, exhibits an intricate series of invaginations (1, 2) that harbor the reaction center (RC) polypeptide subunits. The RCs have been isolated by detergent extraction (for a review, see, for example, reference 3) and are believed to be integral membrane proteins (4). The subunits, designated L, M, and H, are in a 1:1:1 stoichiometry (5). Their molecular weights, determined from an analysis of their amino acid compositions (6, 7) were found to be 28,000, 32,000, and ~34,000, respectively. Each RC contains four bacteriochlorophylls, two bacteriopheophytins, two ubiquinones, and one iron (3). Together with these cofactors, the RC protein accomplishes the conversion of light into electrochemical energy.According to the chemiosmotic hypothesis (8), the topographical organization of the membrane components is fundamental to the directional transfer of protons and electrons and to the coupling of these events to the generation of ATP. Thus, localization of the RC subunits with respect to the membrane should aid in determining their function in the primary charge separation which initiates cyclic electron transfer.Several techniques have been used to investigate the topography of the RC subunits. They include: precipitation with (9), and adsorption of (10), antisera, labeling with antibodies (11-14), radiochemical labeling ( 15-18), photoaffinity labeling (19), and enzymatic digestions (16,18,20). These studies have shown that the RC is an integral membrane protein with the H subunit being exposed on the cytoplasmic side of the membrane. However, no clear concensus has been reached concerning the topography of the other subunits (for a more detailed discussion, see last section of this paper).We used specific antibodies to probe the topography of RCs in the membrane by indirect immunoferritin labeling (21). In this technique, the photosynthetic membrane is first exposed to rabbit antibodies directed against the RC subunits and then to ferritin-conjugated goat antibodies that bind to rabbit IgG. Ferritin is an electron-dense molecule, thereby permitting localization of the binding site by electron microscopy.We performed our initial work on chromatophores, which are closed, inverted membrane vesicles purified from disrupte...

This novel, competitive immunoassay simultaneously detects seven drugs of abuse in urine. A urine sample is placed in contact with lyophilized reagents, the reaction mixture is allowed to come to equilibrium (10 min), and then the whole mixture is applied to a solid phase that contains various immobilized antibodies in discrete drug-class-specific zones. After a washing step, the operator visually examines each zone for the presence of a red bar. The method incorporates present threshold concentrations that are independent for each drug. In the absence of drug or in the presence of drug in quantities less than the threshold concentration, no colored bar is visible. Samples containing drug(s) at or above the threshold concentration cause a red bar to appear for the appropriate drug(s). Positive and negative procedural control zones are incorporated into each determination. The performance of the assay methodology matches that of instrumented immunoassay systems.

A new format for solid-phase immunoassays has been developed in which a monoclonal antibody-coated membrane, incorporated into a cylindrical, disposable device, regulates sample and reagent delivery. We illustrate the method with a two-site, immunoenzymometric assay that can detect human choriogonadotropin at less than 50 int. units/L (4 micrograms/L) in urine and less than 25 int. units/L (2 micrograms/L) in serum and takes less than 5 min to perform. The solid-phase antibody is located in a circular area in the center of the membrane so that in the presence of the hormone, after addition of substrate, a blue enzyme product is generated in this circular area. The high ratio of surface area to volume within the microporous matrix of the membrane assures short diffusion distances and therefore rapid binding of liquid-phase reagents to the solid phase. Pseudo-first-order reaction kinetics describe the binding of antigen to immobilized antibody and the binding of enzyme-labeled antibody to immobilized antigen. The speed and simplicity of this format may facilitate testing for many analytes, both soluble and particulate, as well as serological testing.