Monoclonal antibodies against calcitonin. Characterization and application in light and electron microscopy immunocytochemistry

Twenty-seven monoclonal antibodies (MAbs) to synthetic human calcitonin (CT) were characterized for their reactivities with human CT peptide fragments by dot-blot analysis on nitrocellulose paper. Most of the antibodies bound to the C-terminus and fewer to the mid-region of CT. We have studied thyroid tissue specimens from several animal species after fixation in paraformaldehyde-, glutaraldehyde- or picric acid-containing mixtures and cryostat sectioning or embedment in paraffin or plastic (Epon 812 or Lowicryl 4KM) using this panel of MAbs. The site of antigen-antibody reaction was revealed either by immunoperoxidase, immunoalkaline phosphatase or by silver-enhanced immunogold staining methods. All MAbs were able to localize CT in human, rat and mouse thyroid C cells. Nineteen MAbs recognizing synthetic salmon CT and synthetic [Asu1,7]-eel CT by dot-blot, reacted with chicken ultimobranchial body C cells. One MAb recognizing native porcine CT by dot-blot, stained C cells in hog thyroid. Immunopositivity was confined to the cytoplasm and ultrastructural immunogold labelling demonstrated that cytoplasmic secretory granules were stained. Surgical specimens from human medullary thyroid carcinoma were also analysed for the presence of CT and a variable number of positive cells was found. Furthermore, Congo red-positive areas were shown to react with the MAbs. All conventional staining and immunoabsorption controls were negative. Hence, these MAbs may be suitable for use in routine immunopathological diagnosis of CT-producing tumors and for immunocytochemical localization of the three major CT variants in different animal species.     

抗鹅免疫球蛋白轻链单克隆抗体的鉴定及其应用

免疫球蛋白是机体固有免疫系统的组成部分,是机体防御的第一道防线。本研究对抗鹅免疫球蛋白轻链单克隆抗体进行了特征分析并将其应用到不同免疫试验中用以检测鹅免疫球蛋白。用此单克隆抗体制备的免疫亲和层析柱用以分离血清中的鹅免疫球蛋白;偶联辣根过氧化物酶 (Horseradish peroxidase,HRP) 后的单克隆抗体用作第二抗体来检测鹅特异性抗体。此外,该单克隆抗体可以识别和定位外周血淋巴细胞中的SIg+淋巴细胞。研究表明,该单克隆抗体可在多种条件下检测或分离鹅免疫球蛋白并作为研究鹅体液免疫的有力工具。     

Position of the amino terminus of myosin light chain 1 and light chain 2 determined by electron microscopy with monoclonal antibody

The position of the N terminus of myosin light chain 1 (LC1) and myosin light chain 2 (LC2) of rabbit skeletal muscle was mapped on the myosin head with a monoclonal antibody (SI304), which recognized the amino acid sequence N-trimethylalanyl-prolyl-lysyl-lysyl at the N terminus of LC1 and LC2. The complex of the antibody and myosin was observed by electron microscopy. By selective cleavage of the N terminus of LC1 or LC2 with papain or chymotrypsin, the position of the N terminus of LC1 and LC2 was determined separately. The N terminus of LC2 is located at the head-rod junction. The N terminus of LC1 is 11 nm (+/- 3 nm, standard deviation) from the head-rod junction. This position is near the actin-binding site of the myosin head.     

A monoclonal antibody against the glutaraldehyde-conjugated polyamine, putrescine: application to immunocytochemistry

We developed a mouse monoclonal antibody (mAb; APUT-32, IgG1 subisotype mAb) against putrescine (Put) conjugated to bovine serum albumin using a glutaraldehyde (GA)-sodium borohydride procedure, for applications in immunocytochemistry (ICC). The antibody specificity was evaluated by an ELISA binding test, simulating the ICC of tissue sections. APUT-32 mAb was highly specific to Put, and distinguished alterations in the chemical structure of other polyamine (PA) analogs, showing 3.8% crossreaction with cadaverine, 3.3% with spermidine (Spd), and 2.3% with 1,3-diaminopropane. Comparable results in immunoreactivity of APUT-32 mAb were obtained with the ELISA inhibition test. By the indirect immunoperoxidase method using the APUT-32 mAb, Put-like immunoreactivities were observed in the cytoplasm of HeLa and MCF-7 cell lines fixed with GA in combination with NaBH4 reduction, but almost no immunoreaction was seen in the cytoplasm of the human melanoma BD cell line. On the other hand, the same method but using a previously prepared ASPM-29 mAb, specific for spermine (Spm) and Spd, produced intense immunostaining in the cytoplasm of all the three cell types. The Put-like immunoreaction was completely abolished by absorption of the APUT-32 mAb with 10 microg/ml Put-human serum albumin conjugate prepared using GA and NaBH4. HPLC analysis was also performed for the levels of each of the PAs in the three types of cell, showing that the levels of Put detected were much lower than those of Spm and Spd, and were strikingly different in the three cell lines among which the human melanoma BD cell line contained the lowest levels of Put. These results strongly suggest that APUT-32 mAb reacts specifically with Put in the tumor cells and therefore has the potential as a new tool for elucidating the biological roles of Put in cells and tissues.     

Diimidoesters: Role in electron and light microscopy

Diimidoesters are a class of bifunctional reagents which cross-link by reacting with α- and ϵ-amino groups of proteins. The structure and properties of these reagents as well as their reaction with proteins or cells are described. The importance of the type and concentration of the buffer and of the pH as well as the osmolarity and penetration of these fixatives are discussed. Possible artifacts produced during fixation and the use of diimidoesters in enzyme cytochemistry and immunocytochemistry are also reported. A comparison of the cross-linking and structures obtained by diimidoesters with those obtained by aldehydes is stated.     

A combined light and scanning electron microscopy study

The uppermost Eocene Florissant Formation, Rocky Mountains, Colorado, has yielded numerous insect, vertebrate and plant fossils. Three previous comprehensive palynological studies investigated sections of lacustrine deposits of the Florissant Formation and documented the response of plant communities to volcanic eruptive phases but overall found little change in plant composition throughout the investigated sections. These studies reported up to 150 pollen and spore phenotypes. In the present paper, we used a taxonomic approach to the investigation of dispersed pollen and spores of the Florissant Formation. Sediment samples from the shale units containing macrofossils were investigated using light microscopy (LM) and scanning electron microscopy (SEM). The general picture of the palynoflora is in agreement with previous studies. However, the combined LM and SEM investigation provides important complementary information to previous LM studies. While a fairly large amount of previous pollen determinations could be confirmed, the purported taxonomic affinities of several pollen phenotypes need to be revised. For example, pollen referred to as Podocarpus or Podocarpidites sp. belongs to the Pinaceae Cathaya, Malus/Pyrus actually belongs to Dryadoideae, pollen of the form genus Boehlensipollis referred to as Proteaceae/Sapindaceae/Elaeagnaceae or Cardiospermum belongs to Sapindaceae but not to Cardiospermum, and pollen of Persicarioipollis sp. B with previously assumed affinities to Polygonaceae actually belongs to Thymelaeaceae. Pandaniidites and one type of Malvacipollis cannot be linked with Pandanaceae and Malvaceae. A few taxa are new records for Florissant (Ebenaceae: Diospyros; Mernispermaceae; Trochodendraceae: Tetracentron). In general, SEM investigations complement the LM palynological studies and improve the identification of dispersed pollen and spores and enable integration of data from dispersed fossil pollen into a wide range of comparative morphological, taxonomic, evolutionary, biogeographic and phylogenetic studies.     

A new entomopoxvirus from a cockroach: light and electron microscopy

The cockroach entomopoxvirus caused a chronic infection in cultures of the German cockroach Blattella germanica. Heavily infected specimens showed a reduced mobility. Ellipsoid virus occlusion bodies (8 x 5 to 19 x 12 microm) were found intracellularly in tracheole cells, in the hypodermis, in fat body cells, and in muscles. Several hundred virus particles were integrated in a single occlusion body (OB), their long axis being oriented axially. Ovoid viroids measured 320 x 190 nm and possessed a unilateral, concave core and one lateral body. Starting occlusion, small granules attached to the virus particles which later transformed to a beaded, wavy envelope. An initial halo around the occluded virions disappeared in more central regions of the OB. Virus particles were formed either in a dense cytoplasmic area containing electron-dense viroids, or in a loosely aggregated viroplasm. In the latter, developmental stages were mainly represented by spheres with double membranes enclosing granular material. Spindles and larger crystal-like virus-free inclusion bodies occurred in the cytoplasm. The cytoplasm of infected cells appeared degenerated and the chromatin of the nuclei condensed at the periphery or disintegrated. Taxonomically, the described virus exhibits features of both EPV genus A and EPV genus B. Provisory it is named Blattella germanica EPV (BgEPV). A possible use of the cockroach EPV as a biological control agent is discussed.     

The crater defect in boar spermatozoa: A correlative study with transmission electron microscopy,scanning electron microscopy,and light microscopy

Nuclear vacuoles resembling the “crater defect” described in bull spermatozoa were observed in 14 boars. Both the incidence of the defect and semen quality were monitored with phase contrast microscopy over a three-month period. The percentages of cratered spermatozoa varied widely both among boars and in ejaculates from the same boar taken on different days. The presence of cratered spermatozoa at a level of 5% or more appeared to be associated with low semen quality. The defect was studied with scanning and transmission electron microscopy and was found to consist of nuclear invaginations, about 0.5 μm in diameter, containing some scanty amorphous electron-dense material. In boars showing a high incidence of spermatozoa with crater defects, abnormalities of the acrosome and perforatorium were common.     

Submolecular domains of bovine brain kinesin identified by electron microscopy and monoclonal antibody decoration

Kinesin is a microtubule-activated ATPase thought to transport membrane-bounded organelles along MTs. To illuminate the structural basis for this function, EM was used to locate submolecular domains on bovine brain kinesin. Rotary shadowed kinesin appeared rod-shaped and approximately 80 nm long. One end of each molecule contained a pair of approximately 10 x 9 nm globular domains, while the opposite end was fan-shaped. Monoclonal antibodies against the approximately 124 kd heavy chains of kinesin decorated the globular structures, while those specific for the approximately 64 kd light chains labeled the fan-shaped end. Quick-freeze, deep-etch EM was used to analyze MTs polymerized from tubulin and cross-linked to latex microspheres by kinesin. Microspheres frequently attached to MTs by arm-like structures, 25-30 nm long. The MT attachment sites often appeared as one or two approximately 10 nm globular bulges. Morphologically similar cross-links were observed by quick-freeze, deep-etch EM between organelles and MTs in the neuronal cytoskeleton in vivo. These collective observations suggest that bovine brain kinesin binds to MTs by globular domains that contain the heavy chains, and that the attachment sites for organelles are at the opposite, fan-shaped end of kinesin, where the light chains are located.     

Development and application of a monoclonal antibody against Thiothrix spp.

Historically, methods used to identify Thiothrix spp. in environmental samples have been inadequate because isolation and identification procedures are time-consuming and often fail to separate Thiothrix spp. from other filamentous microorganisms. We described a monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) procedure which was used to identify Thiothrix spp. in wastewater, artesian springs, groundwater, and underwater subterranean samples. The ELISA utilized monoclonal antibody T3511 to a species-specific carbohydrate epitope of Thiothrix spp. No cross-reactions were observed among non-Thiothrix strains consisting of 12 species and nine genera. In field trials, the ELISA identified 100% of 20 biochemically and cytologically confirmed Thiothrix spp.-containing samples with no false positives. Indirect immunofluorescent microscopy utilizing T3511 was effective for wastewater samples but not for those from natural spring water because of background fluorescence in the latter. In addition, electron micrographs of Thiothrix spp. labeled with T3511-biotin-anti-mouse antibody-gold showed that epitope T3511 was intracellular both in laboratory strains and environmental isolates. The minimum level of detection of the ELISA was 0.10 microgram/ml.     

Peroxidase-labeled antibody staining for carcinoembryonic antigen of cytologic specimens for light and electron microscopy

Immunohistochemical staining methods suitable for light and electron microscopic examination of cytologic specimens are described. Application of the methods clearly demonstrated the localization of carcinoembryonic antigen (CEA) in adenocarcinoma cells in body fluids. The use of a peroxidase-labeled antibody method permits rapid penetration of the cells by the antibody, which is not achieved by the peroxidase-antiperoxidase or avidin-biotin-peroxidase-complex staining methods. Since mesothelial and inflammatory cells are negative for CEA, the staining of body fluids for CEA is expected to be an extremely useful tool for the differential diagnosis of adenocarcinoma.     

Peroxidase labelled monoclonal antibody against light chains of human cardiac myosin.

Monoclonal antibody against light chains of human cardiac myosin (MLC) was labelled with horseradish peroxidase. The conjugation was performed by two different methods with glutaraldehyde and periodate respectively. The binding activities of the conjugates were tested by enzyme linked immunosorbent assay (ELISA) on the microtitration plates with immobilized MLC (1-1000 ng per well). A comparison of both methods revealed their universal suitability for the preparation of conjugates as well as their applicability. The use of conjugates shortens the time needed and improves the ELISA method for MLC estimation. Specific advantages of the glutaraldehyde and the periodate method concern diverse details.     

一种新的抗人角蛋白单克隆抗体

In this paper, we reported a novel monoclonal antibody against human keratins, R 6-2-14. The antigen used for immunization was derived from human callus, keratins in which traditionally are classified as "Soft" keratins. However, when we studied the tissue specificity of this antibody, it was found that it only reacted strongly with "Hard" keratins of various mammalian species, but no detectable cross-reactivity with any of the "Soft" keratins. This antibody may provide a useful tool for the study of hair regeneration, nail regeneration, corn pathology and differentiation of mammalian epidermal derivatives.     

Regulation of retinal dopamine biosynthesis and tyrosine hydroxylase activity by light

Dopamine (DA)-containing neurons of the rat retina are apparently activated transsynaptically by photic stimulation. Exposure of dark-adapted rats to light increases retinal DA biosynthesis and metabolism. Associated with the light-evoked increase of DA biosynthesis is a rapid activation of tyrosine hydroxylase (TH), the rate-limiting enzyme of catecholamine biosynthesis. The activation of TH is characterized by an increased affinity of the enzyme for the pteridine cofactor. Because TH in dark-adapted retinas is apparently not saturated with cofactor, the light-evoked increase of affinity is probably responsible for the observed stimulation of DA biosynthesis. Cyclic AMP (cAMP)-dependent protein phosphorylation in vitro activates TH extracted from dark-adapted retinas, and phosphorylation-induced TH activation is very similar and not additive with light-evoked activation of the enzyme. Incubation of viable cell suspensions of dissociated retinas with 8-bromo cAMP also activates TH, which indicates the availability of sufficient cAMP-dependent protein kinase in the proper subcellular compartment to regulate the enzyme in situ. The DA-containing neurons of the rat retina are tonically inhibited in darkness, and evidence is presented that this tonic inhibition involves direct synaptic input to the DA neurons from gamma-aminobutyric acid-containing amacrine cells. The DA-containing neurons are also subject to feedback inhibition through DA receptors, and to modulation by alpha 2-adrenergic receptors.     

First visualization of dopaminergic neurons with a monoclonal antibody to dopamine: a light and electron microscopic study

A monoclonal antibody recently synthesized against dopamine (DA) was tested in rat and mouse brain sections after further treatment by PAP immunocytochemistry at the light and electron microscopic levels. Distribution of DA-immunoreactive cell bodies was examined in the substantia nigra (sn), the ventral tegmental area (vta), and the raphe nuclei. DA-immunoreactive fibers were investigated in two DA projection systems, the striatum and the septum. Many dopaminergic cell bodies were found in the sn and the vta. Some scattered DA neurons were encountered in the pars reticulata of the sn. The dorsal raphe and linearis raphe nuclei displayed sparse immunoreactive neurons and a dense plexus of DA fibers. Immunoreactive fibers were observed in the entire striatum, more dense in the ventral part. In the septum, immunonegative neurons were outlined by thin DA fibers in synaptic contact with their somata or dendrites. According to our observations, this DA monoclonal antibody seems to be a selective and sensitive tool for studying the dopaminergic neuronal circuitry at both histological and ultrastructural level.     

A novel human IgA monoclonal antibody protects against tuberculosis

Abs have been shown to be protective in passive immunotherapy of tuberculous infection using mouse experimental models. In this study, we report on the properties of a novel human IgA1, constructed using a single-chain variable fragment clone (2E9), selected from an Ab phage library. The purified Ab monomer revealed high binding affinities for the mycobacterial α-crystallin Ag and for the human FcαRI (CD89) IgA receptor. Intranasal inoculations with 2E9IgA1 and recombinant mouse IFN-γ significantly inhibited pulmonary H37Rv infection in mice transgenic for human CD89 but not in CD89-negative littermate controls, suggesting that binding to CD89 was necessary for the IgA-imparted passive protection. 2E9IgA1 added to human whole-blood or monocyte cultures inhibited luciferase-tagged H37Rv infection although not for all tested blood donors. Inhibition by 2E9IgA1 was synergistic with human rIFN-γ in cultures of purified human monocytes but not in whole-blood cultures. The demonstration of the mandatory role of FcαRI (CD89) for human IgA-mediated protection is important for understanding of the mechanisms involved and also for translation of this approach toward development of passive immunotherapy of tuberculosis.