Recombinant single-chain antibody peptide conjugates expressed in Escherichia coli for the rapid diagnosis of HIV.

Recombinant single chain Fv (scFv) antibody fragments can form the basis of a rapid, whole-blood diagnostic assay. The scFv described in this study is derived from a monoclonal antibody which has a high affinity for glycophorin A, an abundant glycoprotein on the human red blood cell membrane surface. The prototype reagent built around the scFv was designed to detect, in whole blood samples, the presence of antibodies that have arisen through infection with a foreign organism such as human immunodeficiency virus.

The detection of D-dimer in plasma by enzyme immunoassay: improved discrimination is obtained with a more specific signal antibody.

Most commercial D-dimer enzyme immunoassays employ two antibodies, a fibrin specific capture monoclonal and a less specific antibody cross-reacting with epitopes present on fibrinogen. Two different ELISA systems were compared to test whether this cross-reaction can lead to overestimation of the true levels of cross-linked fibrin derivatives. Both assays used the same D-dimer specific antibody for capture, DD-3B6/22, but utilized signalling antibodies which differed in fibrinogen reactivity.

Automated determination of cross-linked fibrin derivatives in plasma.

Automated assays for the measurement of cross-linked fibrin derivatives in plasma (XL-FbDP) have been developed utilizing latex beads coated with anti-D dimer monoclonal antibody (DD-3B6/22) for both the Cobas Fara Chemistry Centrifugal and the Cobas Mira analysers (Roche, Basle, Switzerland). The analysers were programmed to mix plasma and latex reagent simultaneously and analyse absorbance changes over a 10-15 min period. Results were interpolated by the analyser from a standard curve derived from a polymer of D-dimer.

Rapid whole blood assay for HIV-1 seropositivity using an Fab-peptide conjugate.

A rapid whole blood test has been developed for circulating antibodies to human immunodeficiency virus type 1 (HIV-1), based on agglutination of autologous red blood cells. Evaluation of the test revealed that 100% of seropositive HIV-1 patients (both asymptomatic and AIDS cases) were detected (n = 94) with a specificity of 99.5% in healthy blood donors (n = 596).

The simpliRED D dimer test: a novel assay for the detection of crosslinked fibrin degradation products in whole blood.

A new system for the detection of fibrin degradation products in whole blood has been developed. The test provides a clearly visible agglutination of the patient's red blood cells in the presence of elevated levels of the crosslinked fibrin derivative, D dimer. The test, which uses a bispecific reagent prepared from Fab' fragments of monoclonal antibodies, gives a positive result in 1-2 minutes.

Presence of a substance P-like immunoreactive neurone system from the parabrachial area to the central amygdaloid nucleus of the rat with reference to coexistence with calcitonin gene-related peptide.

An ascending neurone system containing substance P-like immunoreactivity (SPI) from the lateral parabrachial nucleus (PBL) to the central amygdaloid nucleus (AC) was detected. Destruction of the external subdivision of the PBL resulted in a marked ipsilateral reduction of SPI fibres in the AC, which suggests that SPI neurones project mainly ipsilaterally to the AC. This was supported by the findings that injection of biotin-wheatgerm agglutinin into the AC labelled many neurones in the ipsilateral external subdivision of the PBL.

Projection of neurotensin-like immunoreactive neurons from the lateral parabrachial area to the central amygdaloid nucleus of the rat with reference to the coexistence with calcitonin gene-related peptide.

The origin of neurotensin-like immunoreactive (NTI) fibers in the central amygdaloid nucleus (AC) in the rat was examined using indirect immunofluorescence and retrograde tracing combined with immunocytochemistry. Destruction of the external subdivision of the lateral parabrachial nucleus, which contains a group of NTI neurons, resulted in a marked reduction of these fibers in the ipsilateral AC, which suggests that most of these fibers are of extrinsic origin. This was also supported by the finding that injection of fast blue dye into the AC labeled many neurons in the external subdivision of the lateral parabrachial nucleus ipsilaterally, and that simultaneous treatment with antiserum against NT stained some of these neurons.

A latex agglutination assay for D dimer: evaluation and application to the diagnosis of thrombotic disease.

Although latex agglutination assays have been used for some years to diagnose thrombotic disorders, only recently has it been possible to measure specifically the products of fibrin breakdown in the presence of fibrinogen degradation products, by using monoclonal antibodies. We have evaluated a preparation of latex particles coupled to the monoclonal antibody DD-3B6/22, which is specific for cross-linked fibrin degradation products (XDP) and allows accurate discrimination between normal and pathological conditions. Of samples from 515 apparently healthy volunteers, 97.

Calcitonin gene-related peptide-like immunoreactive sensory fibers form synaptic contact with sympathetic neurons in the rat celiac ganglion.

The present study demonstrates synaptic contact between calcitonin gene-related peptide (CGRP)-like immunoreactive axon terminals and sympathetic neurons in the rat celiac ganglion. Our observations suggest that sensory ganglion neurons directly regulate the sympathetic activity via synapses, because CGRP immunoreactive (CGRPI) fibers in this ganglion are supplied by the sensory ganglia.

Distribution and origin of calcitonin gene-related peptide in the rat stomach and duodenum: an immunocytochemical analysis.

We studied the three-dimensional distribution of structures with calcitonin gene-related peptide-like immunoreactivity (CGRPI) in the rat stomach and duodenum, including the origins of these structures, using indirect immunofluorescence in both muscle strips and frozen sections. There was a very dense meshwork of CGRPI fibers in the circular and longitudinal muscle layers, and also in the myenteric and submucous plexuses of the stomach and duodenum. No CGRPI neurons were seen in the stomach, even in rats treated with colchicine; in the duodenum, there was a group of CGRPI cells in the myenteric and submucous ganglia.

Distribution and origins of substance P (SP)-, calcitonin gene-related peptide (CGRP)-, vasoactive intestinal polypeptide (VIP)- and neuropeptide Y (NPY)-containing nerve fibers in the pineal gland of gerbils.

In the pineal gland of gerbils, substance P (SP)-, calcitonin gene-related peptide (CGRP)-, vasoactive intestinal polypeptide (VIP)- and neuropeptide Y (NPY)-containing nerve fibers were demonstrated immunohistochemically. After intrapineal injection of biotin-wheat germ agglutinin, origins of fibers were examined by the combined technique of tracing method and immunohistochemistry. It was confirmed that SP- and CGRP-fibers originated from the trigeminal ganglion, VIP-fibers from the pterygopalatine ganglion and NPY-fibers from the superior cervical ganglion.

Ultrastructural observation of nerve fibers containing both substance P and calcitonin gene-related peptide in the nucleus tractus solitarii of the rat: a combination of immunofluorescence and PAP methods.

Nerve fibers and their axon terminals with substance P (SP)-like and calcitonin gene-related peptide (CGRP)-like immunoreactivity in the nucleus tractus solitarii were ultrastructurally characterized by a combination of immunofluorescent double staining and the PAP method. The axon terminals formed asymmetrical synaptic contacts with other non-reactive neuronal elements (perikarya, dendritic shafts and dendritic spines). Some terminals received synaptic inputs from non-reactive axon terminals.

Calcitonin gene-related peptide in the hepatic and splanchnic vascular systems of the rat.

The distribution and origin of calcitonin gene-related peptide immunoreactive structures in hepatic and splanchnic vasculature of the rat were investigated by the immunofluorescent technique. Calcitonin gene-related peptide immunoreactive fibers were dissociated from compact thick calcitonin gene-related peptide immunoreactive fiber bundles located around the hepatic and splanchnic vasculature, and reached the tunica adventitia of the vasculature. Some fibers penetrated further into the tunica media of the vasculature.

Autoradiographic localization of calcitonin gene-related peptide binding sites in human and rat brains.

125I-calcitonin gene-related peptide (CGRP) binding sites were mapped in the human brain and rat brains by in vitro macroautoradiography, and compared to each other. Binding experiments were made to characterize 125I-CGRP binding on the human and rat brains. Scatchard analysis of saturation experiments from slide-mounted sections of the human and rat cerebellum displayed 125I-CGRP binding sites with a dissociation constant (Kd) of 0.

Ontogeny of calcitonin gene-related peptide and calcitonin in the rat thyroid.

In this immunohistochemical study, the ontogenic development of calcitonin-gene-related peptide (CGRP) in the rat thyroid was investigated and compared with that of calcitonin using the indirect-immunofluorescence method. Parafollicular cells with immunoreactivity to both CGRP and calcitonin first appeared at an early stage of gestation (days 17 and 18) in the central portion of the thyroid. Cells immunoreactive to CGRP and calcitonin had became numerous by gestational day 22.

The presence of a calcitonin gene-related peptide in the olivocochlear bundle in rat.

The origins of calcitonin gene-related peptide immunoreactive (CGRPI) fibers in the cochlea were examined in rats. Parasagittal transection of the brain just medial to the principal sensory trigeminal nucleus resulted in the ipsilateral disappearance of CGRPI fibers in the cochlea, indicating that the origins of these fibers lie in the central nervous system. Next, we used a highly sensitive method combining retrograde tracing and immunohistochemistry to identify the origins of the CGRPI fibers in the cochlea.

Distribution of calcitonin gene-related peptide-containing fibers in the urinary bladder of the rat and their origin.

By use of indirect immunofluorescence, this study demonstrated the presence of calcitonin gene-related peptide-like immunoreactive (CGRPI) fibers in the bladder of the rat. These fibers were abundant in the muscle layer, in which they ran parallel to the muscles, submucosa, and epithelium. No immunoreactive cells were detected.

Localization of calcitonin gene-related peptide in the organ of Corti of the rat: an immunohistochemical study.

The localization of calcitonin gene-related peptide (CGRP)-like immunoreactive (CGRPI) structures in the cochlea was examined in the rat using immunocytochemistry. Numerous CGRPI fibers entered the organ of Corti in the intraganglionic spiral bundle and formed a dense fiber patch at the base of the inner hair cells. Much fewer, but still a significant number of CGRPI fibers were seen at the synaptic region of the outer hair cells.

Calcitonin gene-related peptidergic projection from the parabrachial area to the forebrain and diencephalon in the rat: an immunohistochemical analysis.

We investigated ascending fiber projections of calcitonin gene-related peptide from the parabrachial area to the forebrain and diencephalon in the rat using immunocytochemistry. Destruction of the lateral portion of the dorsal parabrachial area resulted in a marked ipsilateral decrease in the fibers containing calcitonin gene-related peptide in the ventromedial hypothalamic nucleus, indicating that cells containing calcitonin gene-related peptide in the lateral portion of the dorsal parabrachial area projected to the ipsilateral ventromedial hypothalamic nucleus. Destruction of the ventral portion of the parabrachial area resulted in a marked decrease of fibers containing calcitonin gene-related peptide in the bed nucleus of the stria terminalis, the central amygdaloid nucleus and the lateral hypothalamus just medial to the crus cerebri (the far-lateral hypothalamus), and a less marked decrease in the ventromedial thalamic nucleus.

Calcitonin gene-related peptide projection from the ventromedial thalamic nucleus to the insular cortex: a combined retrograde transport and immunocytochemical study.

We employed a highly sensitive combination method of retrograde tracing and immunohistochemistry to identify calcitonin gene-related peptide (CGRP)-containing fiber pathways in the rat from the ventrolateral part of the ventrolateral thalamic nucleus (vl-vl) and the caudal continuation to the insular cortex. Biotin-wheat germ agglutinin (B-WGA) injected into the insular cortex labeled numerous neurons in the vl-vl and the caudal continuation ipsilaterally; simultaneous staining with CGRP antiserum revealed that some of these neurons are CGRP positive.

The calcitonin gene-related peptide-containing fiber projection from the hypothalamus to the lateral septal area including its fine structures.

The afferent source of calcitonin gene-related peptide-like immunoreactive (CGRPI) fibers in the lateral septal area of the rat was sought by using indirect immunofluorescence technique. These fibers decreased markedly on the operated side after the destruction of the area between the anterior hypothalamic nucleus and the lateral hypothalamus where there were a number of CGRPI cells. We also examined the fine structure of CGRPI fibers in the lateral septal area.

Effect of calcitonin gene-related peptide on contraction of striated muscle in the mouse.

We have found ultrastructurally calcitonin gene-related peptide-like immunoreactivity in the axon terminal within the synaptic trough of neuromuscular junction of the mouse. We determined, using pharmacological means, with a phrenic nerve-diaphragm preparation, that this peptide enhances muscle contraction during stimulation of the nerve fibers or direct stimulation of the muscle. This effect is probably brought about via the receptor for this peptide not the acetylcholine receptor.

Light and electron microscopic studies of calcitonin gene-related peptide-like immunoreactive neurons and axon terminals of the nucleus of the tractus solitarius of the rat.

This study was an examination of the ultrastructural characteristic features of calcitonin gene-related peptide (CGRP)-like immunoreactive neurons and their axon terminals in the nucleus of the tractus solitarius of the rat. Some axon terminals were identified as receiving synaptic inputs from non-immunoreactive axon terminals. This may suggest that part, if not all, CGRP containing afferents are affected presynaptically by other afferents.