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 scFv was composed of the antibody heavy-chain variable domain (Vh) joined by a 15 residue linker -(GGGGS)3- to the light-chain variable domain (V1) terminated by either a C-terminal octapeptide tail (FLAG) or a 35 amino acid segment from the gp41 surface glycoprotein of HIV-1. Constructs were cloned into a Escherichia coli expression vector, pHFA, and expressed in a soluble form into culture supernatant. The product retained anti-glycophorin activity which could be detected directly in culture supernatants by ELISA. Furthermore, the scFv-epitope fusion functioned efficiently in the whole blood agglutination assay and was able to distinguish between HIV-1 positive and negative sera.

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. Both assays had high precision (< 5% CV) for values between 100 and 1000 ng/ml and provided clear discrimination between normal samples and samples from patients suffering from the thrombotic diseases, DVT/PE and DIC. The results obtained for XL-FbDP determination with both methods compared well with established methods: a high correlation was obtained with a semi-quantitative manual latex method for both the Fara (r = 0.92) and Mira (r = 0.83) and correlations (r) of 0.81 (Fara) and 0.84 (Mira) were obtained with an enzyme immunoassay (EIA). Correlation between the two automated procedures was high (r = 0.96). The automated method will enable laboratories to provide a rapid and accurate quantitation of XL-FbDP.

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 assay uses an Fab fragment of a monoclonal antibody specifically directed against glycophorin (a transmembrane glycoprotein present on the surface of human red blood cells). This anti-red blood cell Fab is conjugated via the inter-heavy chain cysteines to a synthetic peptide corresponding to the immunodominant epitope of the HIV-1 viral coat protein gp41 (579-613). Addition of this reagent to 10 microliters of whole blood results in the Fab-peptide conjugate coating the red blood cells with peptide. In the presence of circulating antibodies to the HIV-1 peptide, red cell agglutination occurs within 2 min. The sensitivity and specificity of this reagent indicate that it is appropriate for use as a rapid diagnostic test for HIV-1 seropositivity.

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. One monoclonal antibody (RAT-1C3/86) was raised against human red blood cells, and the second (DD-3B6/22) was specific to the crosslinked fibrin derivative, D dimer. Addition of the bispecific reagent to a drop of patient's whole blood resulted in red blood cell agglutination when elevated levels of D dimer were present in the sample. Clinical trials showed sensitivity equivalent to that of current commercial tests. Samples from patients with thrombotic disease states as well as normals were examined. The test was compared with commercial latex agglutination and enzyme immunoassay systems and showed good correlation with the presence of elevated levels of crosslinked fibrin degradation products. This technology represents an advance which allows rapid "on the spot" whole blood analysis, for the diagnosis of thrombotic disorders.

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. Simultaneous staining with antiserum showed that some of these neurones contain SP. Immunohistochemical double-staining revealed that almost all of the SPI neurones in the external subdivision of the PBL contained calcitonin gene-related peptide.

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. Subsequent immunohistochemical staining of alternate sections revealed that many of these NTI neurons were also labeled by calcitonin gene-related peptide antiserum.

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, 97.7% failed to agglutinate the latex; the normal reference interval for XDP determined by enzyme immunoassay was less than 78-320 micrograms/L. The use of different anticoagulants with or without the addition of a protease inhibitor had no significant effects on the results of the latex assay. The latex preparation provides a useful, rapid diagnostic tool for assaying small numbers of samples or as an emergency test.

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. No regional differences were seen in the stomach and duodenum. We found by experimental manipulations that CGRPI fibers in the stomach were exclusively extrinsic in origin; some of such fibers in the duodenum were intrinsic in origin, though most were supplied by CGRPI cells outside the duodenum.

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. This suggests that some, if not all, afferents containing SP and CGRP are affected presynaptically by other afferents.

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. In the tunica adventitia of the vasculature, calcitonin gene-related peptide immunoreactive fibers formed a fine meshwork which could be traced to the branches and arterioles. In the liver, calcitonin gene-related peptide immunoreactive innervation extended from porta hepatis to portal triads, running with branches of the hepatic artery and portal vein. Some calcitonin gene-related peptide immunoreactive fibers separated from thinner calcitonin gene-related peptide immunoreactive fiber bundles in portal triads and coursed into the immediately adjacent parenchyma. In the dorsal spinal ganglia (T8-10, L1), calcitonin gene-related peptide immunoreactive cells accounted for about 60% of the total number of ganglion cells. Bilateral vagotomy just below the diaphragm or bilateral transection of the greater splanchnic nerve resulted in a marked decrease of the number of calcitonin gene-related peptide immunoreactive fibers in the vasculature, whereas a combined operation was followed by the complete depletion of calcitonin gene-related peptide immunoreactive fibers. These results indicate that calcitonin gene-related peptide immunoreactive innervation in hepatic and splanchnic vasculature may have a dual origin. In conclusion, the widespread distribution of calcitonin gene-related peptide immunoreactive fibers in the hepatic and splanchnic vasculature suggest that calcitonin gene-related peptide innervation may be involved in sensory transmission.

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 0.17 nM and 0.11 nM, respectively, and a maximal number of binding sites (Bmax) of 96.8 fmol/mg and 23.0 fmol/mg protein. 125I-CGRP binding was time-dependent, reversible and saturable with high affinity in the brains. Autoradiograms showed a discrete distribution of 125I-CGRP binding sites throughout the brains of human and rat with patterns similar to each other. In the human brain, the highest binding was seen in the cerebellum, inferior olivary nuclear complex, certain parts of the central gray matter, arcuate nuclei of the medulla oblongata and dorsal motor nucleus of the vagus, and densities of CGRP-binding sites were high in the nucleus accumbens, amygdala, tail of the nucleus caudatus, substantia nigra, ventral tegmental area, medial portion of the inferior colliculus, medial pontine nuclei, locus coeruleus, inferior vestibular nucleus, substantia gelatinosa of the spinal trigeminal nucleus, nucleus of the solitary tract and nucleus cuneatus lateralis. In the rat, high densities were found in the hippocampus pars anterior, nucleus accumbens, ventral and caudal portions of the nucleus caudatus-putamen, central and basolateral nuclei of the amygdala, caudal portion of the insular cortex, medial geniculate body, superior and inferior colliculi, certain portions of the central gray matter, locus coeruleus, inferior olivary nuclei, vagal complex, nucleus cuneatus lateralis and cerebellum. In contrast, in both species, most of the cortical areas including the hippocampus, most of the thalamus, and hypothalamus exhibited few binding sites. In addition, high quantities of the binding sites were seen on the pia mater and on walls of blood vessels in the brain and subarachnoidea. These results revealed essentially homologous locations of CGRP binding sites in the human and rat central nervous systems and well corresponding distributions of binding sites and endogenous CGRP-like immunoreactivity.

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. After injection of biotin-wheat germ agglutinin (b-WGA) into the cochlea, CGRPI neurons in the ipsilateral lateral superior olivary nucleus also contained b-WGA granules. These findings indicated that CGRPI efferent fibers are major components of the olivocochlear bundle.

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. After postnatal day 7, CGRP- and calcitonin-immunoreactive (C-IR) cells increased rapidly both in number and in the intensity of their fluorescence. In 14- to 90-day old rats, many intensely immunoreactive cells were distributed in the central portion of the thyroid. The cells immunoreactive to CGRP and to calcitonin had an almost identical ontogenic appearance. In 14-day-old and adult rats, C-IR cells also exhibited CGRP immunostaining, suggesting that these cells simultaneously produce and store CGRP during ontogeny.

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. Since no immunoreactive cells were seen in the organ of Corti and spinal ganglion, these fibers may be one of the major components of the olivocochlear bundles originated from the superior olivary complex.

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. This means that there are projections from cells containing calcitonin gene-related peptide in the ventral portion of the parabrachial area to the first three regions just mentioned, and to some extent to the last.

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.